Methods of NAD-dependent deacetylation of a lysine residue in a protein

ABSTRACT

Methods of NAD-dependent of at least one lysine residue in an acetylated protein are disclosed. The methods include combining the acetylated protein with an isolated Sir2 protein or fragment that includes a core domain of the Sir2 protein. The Sir2 protein or fragment of the Sir2 protein can include a human Sir2 protein or a fragment of a human Sir2 protein.

RELATED APPLICATIONS

This application is a continuation and claims priority under 35 U.S.C.§120 to U.S. application Ser. No. 09/461,580, filed Dec. 15, 1999, nowU.S. Pat. No. 7,452,664. The disclosure of this application isincorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant number R01AG011119 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Aging involves progressive and irreversible loss of cellular processesand physiological functions that ultimately increase the likelihood ofdeath. Molecular correlates of aging, including an increase inchromosomal structural abnormalities, the frequency of single-strand DNAbreaks, a decline in DNA methylation, and a loss of DNA telomericsequences, have been described in a range of eukaryotic organisms frommammals, such as humans, to unicellular organisms, such as yeast.

Although several mechanisms have been postulated as mediators of aging(e.g., somatic mutation theory, error catastrophe theory, intrinsic DNArearrangement theory), none have led to interventions or therapies toslow aging and increase life span. In humans, declining health in agingindividuals has a significant impact on the cost and implementation ofgeriatric health care.

Thus, there is a need to identify agents which alter (e.g., agonize,antagonize) the level of substrates and cellular mediators associatedwith the aging process. The identification of such agents is importantin the development of specific and effective treatment regimens todecrease aging or increase the life span of a cell or an organism, andto further define pathways which lead to aging in a cell or organism.

SUMMARY OF THE INVENTION

The present invention relates to methods of altering the NAD-dependentacetylation status of proteins, e.g., histone proteins, identifyingagents which alter the NAD-dependent acetylation status of proteins,e.g., histone proteins, identifying agents which altermono-ADP-ribosylation of nuclear proteins, alter aging or alter lifespan, as well as to methods of altering mono-ADP-ribosylation of nuclearproteins, methods of altering aging of a cell or organism, and methodsof altering life span of a cell or organism. In preferred embodiments,the invention relates to methods of identifying agents which alter theNAD-dependent acetylation status of histone proteins by altering theactivity of Sir2, increase mono-ADP-ribosylation of nuclear histoneproteins, decrease aging or increase life span in a cell or organism, aswell as to methods of increasing mono-ADP-ribosylation, decreasing agingor increasing life span of a cell or organism. In preferred embodiments,increasing mono-ADP-ribosylation or NAD-dependent deacetylation ofhistone proteins, decreasing aging or increasing life span of a cell ororganism comprise administering to the cell or organism Sir2 or amono-ADP-ribosyltransferase or an agonist of Sir2 and/ormono-ADP-ribosyltransferase activity, and combinations thereof.

In one embodiment, the method of the invention is a method of alteringthe NAD-dependent acetylation status of at least one amino acid residuein a histone protein by altering the activity of a Sir2 protein or aSir2-like protein. The histone protein can be selected from the groupconsisting of a H2B, H3 or H4 histone protein. In a preferredembodiment, the amino acid residue is a lysine amino acid residue. In aparticularly preferred embodiment, the lysine amino acid residue islysine 9 and/or lysine 14 of a H3 histone protein and/or lysine 16 of aH4 histone protein. The NAD-dependent acetylation status is removal ofan acetyl group and/or addition of an acetyl group.

In a preferred embodiment, NAD-dependent acetylation status of thehistone protein is altered by altering the activity of Sir2α protein. Inanother embodiment, the NAD-dependent acetylation status is altered byaltering the activity of a mutant Sir2α protein selected from the groupconsisting of G253A, G255A, S257A, I262A, F265A, R266A, G270A, P285A,T336A, H355A, Thr-261, Iso-271, Arg-275, Asn-345 or Asp-347.

In another embodiment, the invention relates to a method of identifyingan agent which alters the activity of a Sir2 protein or a Sir2-likeprotein by assessing the NAD-dependent acetylation status of at leastone amino acid in a histone protein, comprising combining the histoneprotein, the Sir2 protein or the Sir2-like protein, NAD or a NAD-likecompound and the agent to be tested, thereby producing a combination;detecting the NAD-dependent acetylation status of an amino acid in thehistone protein; and comparing the NAD-dependent acetylation status inthe presence of the agent to be tested with the NAD-dependentacetylation status of the amino acid in the histone protein in theabsence of the agent to be tested, wherein a difference in theNAD-dependent acetylation status of the amino acid of the histoneprotein between the presence of the agent and the absence of the agentindicates that the agent alters the NAD-dependent acetylation status ofat least one amino acid of the histone protein.

Another aspect of the invention relates to a method of identifying anagent which alters life span of a cell by assessing the NAD-dependentacetylation status of at least one amino acid in a histone protein,comprising combining the histone protein, a Sir2 protein or Sir2-likeprotein, NAD or a NAD-like compound and the agent to be tested, therebyproducing a combination; detecting the NAD-dependent acetylation statusof an amino acid in the histone protein; and comparing the NAD-dependentacetylation status in the presence of the agent to be tested with theacetylation status of the amino acid in the histone protein in theabsence of the agent to be tested, wherein a difference in theacetylation status of the amino acid of the histone protein between thepresence of the agent alters the life span of the cell. The inventionfurther relates to administering the agents identified by the method toa cell and assessing the NAD-dependent acetylation status of at leastone amino acid in a histone protein of the cell.

In a further embodiment, the invention relates to a method ofidentifying an agent which alters the activity of a Sir2 protein or aSir2-like protein by assessing the NAD-dependent acetylation status ofat least one amino acid in a histone protein, comprising combining thehistone protein, the Sir2 protein or the Sir2-like protein, NAD or aNAD-like compound and the agent to be tested, thereby producing acombination; detecting the NAD-dependent acetylation status of an aminoacid in the histone protein; and comparing the NAD-dependent acetylationstatus in the presence of the agent to be tested with the NAD-dependentacetylation status of the amino acid in the histone protein in theabsence of the agent to be tested, wherein a difference in theNAD-dependent acetylation status of the amino acid of the histoneprotein between the presence of the agent and the absence of the agentindicates that the agent alters the NAD-dependent acetylation status ofat least one amino acid of the histone protein.

In yet another embodiment, the invention relates to a method ofidentifying an agent which alters aging of a cell by assessing theNAD-dependent acetylation status of at least one amino acid in a histoneprotein, comprising combining the histone protein, a Sir2 protein orSir2-like protein, NAD or a NAD-like compound and the agent to betested, thereby producing a combination; detecting the NAD-dependentacetylation status of an amino acid in the histone protein; andcomparing the NAD-dependent acetylation status in the presence of theagent to be tested with the acetylation status of the amino acid in thehistone protein in the absence of the agent to be tested, wherein adifference in the acetylation status of the amino acid of the histoneprotein between the presence of the agent alters aging of the cell. Inone embodiment, the agent increases aging of the cell. In anotherembodiment, the agent decreases aging of the cell.

In another embodiment, the invention relates to a method of altering theNAD-dependent acetylation status of at least one amino acid residue in ahistone protein comprising combining the histone protein, a Sir2 proteinor Sir2-like protein and a NAD or a NAD-like compound.

In yet another embodiment, the methods of the invention include methodsfor identifying an agent which alters mono-ADP-ribosylation of a nuclearprotein in a cell or an organism, comprising combining a cell ororganism and an agent to be tested; determining a level ofmono-ADP-ribosylation of a nuclear protein in the cell or in one or morecells of the organism; and comparing the level of mono-ADP-ribosylationin the presence of the agent with the level of mono-ADP-ribosylation ofthe nuclear protein in the absence of the agent. A difference in thelevel of mono-ADP-ribosylation of the nuclear protein in the presence ofthe agent as compared with in the absence of the agent indicates thatthe agent alters mono-ADP-ribosylation of the nuclear protein. In apreferred embodiment, the agent is an agonist of mono-ADP-ribosylation.In another embodiment, the agent is an antagonist ofmono-ADP-ribosylation.

In another embodiment, the invention relates to methods for identifyingan agent which alters life span of a cell or an organism, comprisingcombining a cell or organism and an agent to be tested; determining alevel of mono-ADP-ribosylation of a nuclear protein in the cell or inone or more cells of the organism; and comparing the level ofmono-ADP-ribosylation in the presence of the agent with the level ofmono-ADP-ribosylation of the nuclear protein in the absence of theagent. A difference in the level of mono-ADP-ribosylation of the nuclearprotein in the presence of the agent as compared with in the absence ofthe agent indicates that the agent alters mono-ADP-ribosylation of thenuclear protein, wherein agents which alter mono-ADP-ribosylation ofnuclear proteins are agents which alter life span of a cell or anorganism. In a preferred embodiment, the agent is an agonist ofmono-ADP-ribosylation. In another embodiment, the agent is an antagonistof mono-ADP-ribosylation.

The invention also relates to a method of identifying an agent whichincreases life span of a cell or an organism, comprising the steps ofcombining a cell or organism and an agent to be tested; determining alevel of mono-ADP-ribosylation of a nuclear protein in the cell or inone or more cells of the organism in the presence of the agent and inthe absence of the agent; identifying an agent which increasesmono-ADP-ribosylation of a nuclear protein; administering said agent toa cell or an organism; and determining the life span of said cell,wherein an agent which increases the life span of said cell or organismrelative to the mean life span of said cell or organism or relative tothe life span of said cell or organism in the absence of the agent is anagent which increases life span of a cell or organism.

The invention also relates to a method of identifying an agent whichdecreases life span of a cell or an organism, comprising the steps ofcombining a cell or organism and an agent to be tested; determining alevel of mono-ADP-ribosylation of a nuclear protein in the cell or inone or more cells of the organism in the presence of the agent and inthe absence of the agent; identifying an agent which decreasesmono-ADP-ribosylation of a nuclear protein; administering said agent toa cell or an organism; and determining the life span of said cell,wherein an agent which decreases the life span of said cell or organismrelative to the mean life span of said cell or organism or relative tothe life span of said cell or organism in the absence of the agent is anagent which decreases life span of a cell or organism.

Also encompassed by the present invention is a method of identifying anagent which alters aging of a cell, comprising the steps of combining acell and an agent to be tested; determining a level ofmono-ADP-ribosylation of a nuclear protein in the cell; and comparingthe level of mono-ADP-ribosylation in the presence of the agent with alevel of mono-ADP-ribosylation of the nuclear protein in the absence ofthe agent to be tested. A difference in the level ofmono-ADP-ribosylation of the nuclear protein between the presence of theagent and the absence of the agent indicates that the agent alters agingof the cell. In particular embodiment, the agent decreases aging of thecell. In another embodiment, the agent increases aging of the cell.

Another aspect of the invention includes a method of increasing the lifespan of a cell, comprising administering to the cell an effective amountof an agent which increases mono-ADP-ribosylation of a nuclear protein.The agent to be administered is identified by a method comprising thesteps of combining a cell and an agent to be tested; determining a levelof mono-ADP-ribosylation of a nuclear protein in the cell; and comparingthe level of mono-ADP-ribosylation in the presence of the agent with alevel of mono-ADP-ribosylation of the nuclear protein in the absence ofthe agent to be tested, wherein in the presence of the agent there is anincrease in the level of mono-ADP-ribosylation of the nuclear protein.

The invention also relates to a method of decreasing aging of a cell,comprising administering to the cell an effective amount of an agentwhich increases mono-ADP-ribosylation of a nuclear protein. The agent tobe administered is identified by a method comprising the steps ofcombining a cell and an agent to be tested;

determining a level of mono-ADP-ribosylation of a nuclear protein in thecell; and

comparing the level of mono-ADP-ribosylation in the presence of theagent with a level of mono-ADP-ribosylation of the nuclear protein inthe absence of the agent to be tested, wherein in the presence of theagent there is an increase in the level of mono-ADP-ribosylation of thenuclear protein.

The invention further pertains to a method of increasing the life spanof a cell or an organism comprising administering to the cell ororganism a mono-ADP-ribosyltransferase or an agonist ofmono-ADP-ribosyltransferase activity in an amount effective to increasethe life span of the cell or organism.

In yet another embodiment, the present invention pertains to a method ofincreasing the life span of a cell or organism comprising administeringto the cell or organism an agonist of mono-ADP-ribosylation of histoneprotein H2B in an amount effective to increase the life span of the cellor organism.

Another embodiment of the invention pertains to a method of decreasingaging of a cell or organism comprising administering to the cell ororganism a mono-ADP-ribosyltransferase or an agonist ofmono-ADP-ribosyltransferase activity in an amount effective to decreaseaging of the cell or organism.

Also encompassed by the present invention is a method of decreasingaging of a cell or organism comprising administering to the cell ororganism an agonist of mono-ADP-ribosylation of histone protein H2B inan amount effective to decrease aging of the cell or organism.

The invention also relates to a method of inhibiting the formation,replication and/or accumulation of rDNA circles in a cell comprisingadministering to the cell a mono-ADP-ribosyltransferase or an agonist ofmono-ADP-ribosyltransferase activity in an amount effective to inhibitthe formation, replication and/or accumulation of rDNA circles.

Another aspect of the invention is a method for decreasing recombinationbetween rDNA in a cell comprising administering to the cell amono-ADP-ribosyltransferase or anagonist of mono-ADP-ribosyltransferaseactivity in an amount effective to decrease recombination between rDNA.

In a preferred embodiment, the nuclear protein in the methods of thepresent invention is selected from the group consisting of histoneproteins H2A, H2B and H3. In particular, the mono-ADP-ribosylation ofnuclear proteins is performed by the core domain of Sir2, the Sir2protein, a fragment of the Sir2 protein (e.g., SEQ ID NOS: 1, 2, 4, 9,10, 11, 12, 14, 19, 20, 21 or 26), or anagonist of Sir2.

In one embodiment of the methods described herein, the cell is a yeastcell. In another embodiment, the cell is a mammalian cell.

The present invention also provides an isolated murine Sir2 protein,preferably the murine Sir2α protein (SEQ ID NO: 26) and the nucleic acidsequence encoding the Sir2α (SEQ ID NO: 25).

The invention also pertains to isolated nucleic acid molecules of murineSIR2 genes operably linked to a regulatory sequence and methods ofpreparing Sir2 proteins by culturing the recombinant host cellscomprising isolated nucleic acid molecules of murine SIR2 genes.

Also encompassed by the present invention, are antibodies, orantigen-binding fragments thereof, which selectively bind a murine Sir2proteins or a murine Sir2-like protein.

The invention described herein provides novel methods to readilyidentify agents that alter the NAD-dependent acetylation status ofnuclear proteins and/or the mono-ADP-ribosylation of a nuclear protein;and to utilize the identified agents to alter NAD-dependent acetylation,ADP-ribosylation, aging and increase life span. For example, theinvention provides unique and efficient ways to forestall senescence andextend life in cells and organisms.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a diagrammatic representation of the phylogenetic tree ofyeast and murine Sir2 protein core domains.

FIG. 1B is a Northern blot analysis of the tissue distribution of murineSIR2α, β, and γ mRNA transcripts.

FIG. 2A is the deduced amino acid sequence of mSir2α (SEQ ID NO: 1) withthe predicted nuclear localization signal underlined.

FIG. 2B is a Western blot of the mSir2α protein (120 kD)immunoprecipitated (IP) from cell extracts of murine NIH3T3 cells and invitro translated (IVF) probed with a polyclonal antibody against theN-terminal 131 amino acids of the protein (αmSir2α).

FIG. 2C is an alignment of the evolutionarily conserved core domains ofyeast (ySir2, GENBANK® Accession Nos: X01419, M21316, SEQ ID NO: 2;yHST1, GENBANK® Accession Nos: U39041, L47120, SEQ ID NO: 3); murine(mSir2α, SEQ ID NO: 4) and Salmonella (CobB, GENBANK® Accession No:U89687, SEQ ID NO: 5) Sir2 proteins. Identical amino acids are boxed. Aputative NAD binding cleft is indicated by asterisks. Less conservedamino acids are shaded.

FIG. 2D is a schematic alignment of the core domain of mSir2α, ySir2pand CobB proteins.

FIG. 3 depicts the immunocytochemical localization of mSir2α in mouseNIH3T3 cells (A, D, G, and J). Cells were counterstained with DAPI (B,E, H, and K). Nucleoli (E), telomeres (H), and centromeres (K) werevisualized using an anti-human nucleolar antibody (ANA-N), telomericFISH, and an anti-human centromeric antibody (ANA-C), respectively.Merged images are shown in C, F, I, and L.

FIG. 4A is a COOMASSIE® blue stained gel of 6×His-tagged recombinantyeast (r-y Sir2p) and murine (r-m Sir2α) Sir2 proteins purified withNi-NTA agarose under native conditions. Arrowheads indicate eachfull-length protein.

FIG. 4B depicts the results of ADP-ribosylation of histone proteins H1,H2A, H2B, H3 and H4 by recombinant r-ySir2p and r-mSir2α.

FIG. 4C illustrates ADP-ribosyltransferases cleaving nicotinamide (Nam)from NAD⁺ (Ade-Rib-P*) creating a linkage between ribose (Rib) andhistone proteins.

FIG. 4D illustrates the dose-dependent modification of H2B and H3 byrecombinant yeast r-ySir2p and murine r-mSir2α proteins.

FIG. 4E illustrates the effects of snake venom phosphodiesterase (SVP)on the removal of radiolabeled ³²P-NAD from Sir2-modified H2B and H3.

FIG. 4F depicts the effects of mono-ADP-ribosylation inhibitors(novobiocin and coumermycin A1), but not poly-ADP-ribosylationinhibitors (3-aminobenzamide (3-ABA) and benzamide), on mSir2αADP-ribosylation of H2B.

FIG. 4G depicts the mono-ADP-ribosyltransferase activity of mSir2αimmunoprecipitated by an antiserum to mSir2α (αmSir2α) from NIH3T3 wholecell extracts.

FIG. 5A depicts the amino acid sequences of the N-terminal tails of H3(SEQ ID NO: 6) and H4 (mono AC, SEQ ID NO: 7, tetra Ac, SEQ ID NO: 8)peptides. The asterisks depict the site of acetylation (mono Ac, tetraAc) to generate peptides with and without acetylated lysines. Human H3GENBANK® Accession No: M26150, mouse H3 GENBANK® Accession Nos: M23459,32460, 32461, 32462. Human H4 GENBANK® Accession Nos: M60749, M16707,mouse H4 GENBANK® Accession No: U62672.

FIG. 5B depicts the effects of mSir2α and ySir2p on diacetylated H3peptide (diAc), but not unacetylated H3 (unAc). A bracket, an arrowhead,and an arrow at right indicate modified peptides, hydrolyzed and/ordecomposed products of ³²P-NAD, and unhydrolyzed ³²P-NAD, respectively.

FIG. 5C illustrates mSir2α modification of Lys16-acetylated H4 peptide(Ac16). Unacetylated (unAc), monoacetylated (Ac5, Ac8, Ac12, or Ac16),and tetraacetylated (tetraAc) peptides were added in the reactions withthe control eluate (pET) or the mSir2α, respectively.

FIG. 6A depicts the highly conserved amino acid residues in the coredomain of mSir2α (SEQ ID NO: 9) and ySir2 (SEQ ID NO: 10) proteins.Identical amino acids are boxed. Arrowheads depict the amino acidresidues used to generate mutant Sir2 proteins which were evaluated forADP-ribosylation activity.

FIG. 6B depicts the ADP-ribosylation activities of mSir2α proteins(Wildtype, WT; vector alone, pET; mutants G253A, G255A, S257A, I262A,F265A, R266A, G270A, P285A, T336A, H355A) on histones H2B and H3.

FIG. 6C illustrates the quantitation of the ADP-ribosylation activitiesof the mSir2α mutants (G253A, G255A, S257A, I262A, F265A, R266A, G270A,P285A, T336A, H355A) compared to wild type (WT) and control pET.

FIG. 6D illustrates the in vivo transcriptional repression by the mSir2αwildtype and mutant core domain fused to the GAL4 DNA binding domain(DBD).

FIG. 7 is a diagrammatic representation of a model of Sir2-mediatedmodification of histone proteins H3 and H4.

FIGS. 8 a, 8 b, 8 c, 8 d, 8 e, 8 f, and 8 g illustrate the in vitrodeacetylation of the H3 peptide (residues 1-20) di-acetylated at lysines9 and 14 by recombinant yeast Sir2p.

FIG. 8 a is a COOMASSIE® blue-stained SDS-PAGE gel of purifiedrecombinant yeast (rSir2p) protein, mouse Sir2 (mSir2α) proteins andvector controls. Full length proteins are indicated by dots.

FIGS. 8 b, 8 c, 8 d, 8 e and 8 f are HPLC chromatograms showingabsorbance at 220 nm of products of deacetylation assays with yeastSir2p and the indicated concentrations of NAD. The efficiencies of thereactions are calculated from the areas under peaks 1, 2, and 4.

FIG. 8 g is a schematic of contents of peaks 1-5 shown in chromatograms.

FIGS. 9 a, 9 b, 9 c and 9 d are electron-spray mass spectroscopy ofpeaks 3-5 of HPLC chromatogram.

FIGS. 9 a and 9 b depict peak 3 of the HPLC chromatogram of thedeacetylase reaction at 1 mM NAD. The molecular weights of the twoproducts in peak 3 correspond to the starting peptide (2370) and thedoubly deacetylated dimer (2-times the molecular weight of the startingpeptide, 4740, minus the difference between the molecular weight of twoacetyl moieties versus two hydrogens, 84, yielding 4656).

FIG. 9 c Peak 4 of the same reaction. The molecular weight of peak 4(4698) corresponds to singly deacetylated dimer peptide.

FIG. 9 d Peak 5 of the same reaction corresponds to dimer peptide(molecular weight 4740).

FIGS. 10 a, 10 b, 10 c, 10 d, 10 e and 10 f are the amino-terminalsequencing of peaks 4 and 5 of the deacetylase reaction at 1 mM NAD.Peaks 4 and 5 were subjected to sequencing by Edmann degradation.Chromatograms at positions 9, 14, and 18 are shown. In peak 4, about 23%of Lys9 (FIG. 10 a) and 27% of Lys 14 (FIG. 10 b) were deacetylated. Inpeak 5, both Lys 9 and 14 are essentially all acetylated (FIG. 10 d andFIG. 10 e). The unacetylated Lys18 of both peaks 4 and 5 are shown forcomparison (FIG. 10 c and FIG. 10 f). The peak to the right of theacetylated lysine at position 14 corresponds to alanine, which is apreview of Ala 15.

FIGS. 11 a, 11 b, 11 c, 11 d and 11 e illustrate the effects ofinhibitors on the deacetylase and putative ADP-ribosyltransferaseactivities of recombinant Sir2p (rSir2p). The HPLC chromatograms of thereactions in the presence of solvent only (FIG. 11 a), 400 nM TSA (FIG.11 b) and 200 μM coumermycin A1 (Coumer) (FIG. 11 e) are shown. Thecalculated efficiencies of the reactions are indicated. The effect of200 μM coumermycin A1 on ADP-ribosylation of the intact histone H3 (FIG.11 c) and the H3 peptide (FIG. 11 d) are examined on SDS-PAGE and TLC,respectively. pET corresponds to the vector control.

FIG. 11 d shows the ADP-ribosylated peptide indicated by a bracket on alonger exposure (left), and NAD and its hydrolysed product are indicatedby an arrow and an arrowhead on a shorter exposure, respectively(right).

FIGS. 12 a, 12 b, and 12 c show the deacetylation activity of the mouseSir2p homolog, mSir2α.

FIG. 12 a is the amino acid sequence of mSir2α (SEQ ID NO: 1). Theevolutionarily conserved core domain of mSir2α is boxed.

FIG. 12 b shows the phylogenetic tree of yeast and mouse Sir2 coredomains. The core domain sequences of three mouse homologs termed α, β,and γ are compared on CLUSTAL X and NJPLOT programs to generate thephylogenetic tree.

FIG. 12 c illustrates the HPLC chromatogram of the product ofdeacetylation assay with 10 μg of recombinant mSir2α protein at 1 mMNAD. The calculated efficiency of the reaction is indicated.

FIGS. 13 a, 13 b, 13 c, 13 d and 13 e illustrate the effects of NADderivatives on the Sir2p deacetylation activity of the H3 peptide. TheHPLC chromatograms of the reactions with no NAD derivatives (FIG. 13 a)and 1 mM concentration of NAD (FIG. 13 b), NADH (FIG. 13 c), NADP (FIG.13 d), and NADPH (FIG. 13 e) are shown. The calculated efficiencies ofthe reactions are also indicated.

FIGS. 14 a, 14 b and 14 c depict the putative ADP-ribosylationactivities of Sir2 protein core domain mutants.

FIG. 14 a is the amino acid sequence of the core domains of ySir2p (SEQID NO: 11), mSir2α (SEQ ID NO: 12) and CobB (SEQ ID NO: 13) aligned andsix highly conserved residues, indicated by arrowheads, were mutated toalanine.

FIG. 14 b shows the 6×His tagged versions of wildtype ySir2p (wt) andthe six mutant Sir2p (Thr-261, Gly-270, Iso-271, Arg-275, Asn-345,Asp-347) and a vector control (vector) expressed in E. coli, purifiedover a Nickel-NTA column and analyzed on a 7% polyacrylamide SDS gel toassess expression levels.

FIG. 14 c depicts the ability of ySir2p and mutant Sir2p proteins tomodify histone H3 with ³²P labeled NAD.

FIGS. 15 a, 15 b, 15 c, 15 d, 15 e, 15 f, 15 g and 15 h illustrate theeffects of mutations in ySir2p on NAD dependent deacetylation of H3. Thepeptides were run on HPLC and the chromatograms based on the absorptionat 220 nm recorded. The efficiency of reactions were determined bysumming the area under the appropriate peaks.

FIGS. 16 a, 16 b, 16 c and 16 d illustrate the effect of mutations ondeacetylation in vitro and silencing in vivo. Wildtype (wt) and mutantSir2 (Thr-261, Gly-270, Iso-271, Arg-275, Asn-345) were integrated intosir2Δ W303R strains.

FIG. 16 a is a Western blot of 25 μg of whole cell extract fromwildtype, sir2Δ and Sir2 mutants probed with Sir2 antibody. The upperband corresponds to Sir2p and a lower background band is included as aloading control.

FIG. 16 b illustrates silencing at HMLα by mating the strains withtester strains of the opposite mating type and monitoring growth ofdiploids on selective media.

FIG. 16 c illustrates telomere-silencing by the ability of strains withtelomeric URA3 to grow on media containing 5-FOA, which is toxic whenURA3 is expressed, but harmless when URA43 is silenced.

FIG. 16 d illustrates rDNA silencing was monitored in a strain with theADE2 marker located within the rDNA array. RPD3 was disrupted to enhancesilencing differences between the wildtype and sir2Δ strains. sir2mutant strains were spotted on complete and -ade media to monitoreffects on silencing.

FIGS. 17 a, 17 b and 17 c illustrate the effect of mutations in Sir2 onrDNA recombination and yeast life span.

FIG. 17 a illustrates W303R with ADE2 at rDNA tested for rDNArecombination rates by counting the frequency of loss of the marker inthe first generation after plating.

FIG. 17 b illustrates the life span of wildtype, sir2Δ, and mutants 261,270, and 275 measured in hmlAE strains where the a/α effects areeliminated.

FIG. 17 c illustrates the life span of wildtype, sir2Δ, and mutants 261and 270 measured in HML+ strains as a sensitive test of a/α silencing.

FIG. 18 is a summary of in vitro and in vivo data regardingADP-ribosyltransferase, deacetylase, HM silencing, telomere silencing,rDNA silencing, rDNA recombination and mean life span for Sir2 wildtypeand mutant Sir2 proteins (Thr-261, Gly-270, Iso-271, Arg-275, Asn-345,Asp-347).

FIG. 19 depicts the amino acid sequence alignment of the core domain ofySir2 (SEQ ID NO: 14), yHST1 (SEQ ID NO: 15), yHST2 GENBANK® AccessionNO: U39063, (SEQ ID NO: 16), yHST3 GENBANK® Accession No: U39062, (SEQID NO: 17), yHST4 GENBANK® Accession No: NC_(—)001136 (SEQ ID NO: 18),mSir2alpha (mSir2α, SEQ ID NO: 19), mSir2beta (mSir2β, SEQ ID NO: 20),mSirg (mSir2γ, SEQ ID NO: 21), and deduced amino acid sequences ofSir2-like core domains (GENBANK® Accession No: A1465098, SEQ ID NO: 22;GENBANK® Accession No: A1465820, SEQ ID NO: 23; GENBANK® Accession No:A1466061, SEQ ID NO: 24).

FIG. 20 depicts the effects of multiple copies of Sir2 on yeast lifespan.

FIGS. 21 a, 21 b, 21 c and 21 d depict the nucleotide sequence (GENBANK®Accession No: AF214646, SEQ. ID NO: 25) and amino acid sequence (SEQ IDNO: 26) of murine Sir2α.

FIG. 22 depicts the nucleotide sequence (SEQ ID NO: 34) and amino acidsequence (SEQ ID NO: 35) of an EST cDNA clone 557657 (Genome Systems,Inc.).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the discovery that Sir2 proteins, which play apart in the life span control mechanisms in eukaryotic cells, alter theNAD-dependent acetylation status of histone proteins andmono-ADP-ribosylate nuclear proteins. In particular, the inventionpertains to the discovery that Sir2 (e.g., murine Sir2α) alters theNAD-dependent acetylation status (e.g., removes and/or adds an acetylgroup) of histone proteins (e.g., histone proteins H2B, H2A, H3 and/orH4) and mono-ADP-ribosylates histone proteins. As a result of thisdiscovery, methods for identifying agents (e.g., agonists) which alterthe NAD-dependent acetylation status of nuclear proteins, such ashistone proteins, and/or their mono-ADP-ribosylation are available toprovide methods and compositions to alter (e.g., slow) aging and alter(e.g., increase) life span of cells and organisms.

The present invention relates to a method of altering the NAD-dependentacetylation status of at least one amino acid residue of a histoneprotein by altering the activity of a Sir2-protein or Sir-2 likeprotein. In a particular embodiment, the NAD-dependent acetylationstatus of H3 and/or H4 is altered. Specifically encompassed by theinvention is the alteration of lysine residues in the N-terminus of H3(e.g., lysine 9 and/or 14) and H4 (e.g., lysine 16).

“NAD-dependent” as used herein refers to a requirement for NAD(nicotinamide adenine dinucleotide) compound in a reaction. The reactioncan performed or take place in the presence (e.g., in vivo) or theabsence (e.g., in vitro) of cells. A “NAD-like compound” is also withinthe scope of the invention and refers to a compound (e.g., a syntheticor naturally occurring chemical, drug, protein, peptide, small organicmolecule) which possesses structural similarity (e.g., adenine, riboseand phosphate groups) or functional similarity (e.g., oxidation ofsubstrates, NAD-dependent deacetylation of histone proteins). Forexample, NAD-like compounds can be NADH, NADP, NADPH, non-hydrolyzableNAD and fluorescent analogs of NAD (e.g., 1, N6-etheno NAD).

The term “NAD-dependent acetylation status” refers to the requirement ofNAD to either transfer (also referred to herein as the addition) orremove (also referred to herein as deacetylation) at least one acetylgroup (e.g., CH₃CO—) to a substrate having OH or NH₂ groups (e.g., atleast one amino acid residue of a histone protein such as H2B, H2A, H3and/or H4). Thus, “acetylation status” can be either acetylation ordeacetylation of a substrate.

In a particular embodiment, the amino acid residue who acetylationstatus is altered is a basic amino acid (e.g., lysine, arginine,histidine). In a preferred embodiment, the acetylation status lysineresidues of histone proteins H1, H2A, H2B, H3 or H4 are altered. In amore preferred embodiment, the acetylation status of lysine residues atthe amino terminus of H3 and H4 is altered In particular, lysine 9and/or lysine 14 of H3 and/or lysine 16 of H4. Any suitable amino acidresidue (e.g. having OH or NH₂ groups) capable of undergoing analteration in acetylation status in a cell and/or an animal or under invitro conditions (e.g., outside a cell or an animal) is within the scopeof the invention.

The invention also relates to a method of identifying an agent whichalters the activity of a Sir2 protein or a Sir2-like protein byassessing the NAD-dependent acetylation status of at least one aminoacid in a histone protein, comprising combining the histone protein, theSir2 protein or the Sir2-like protein, NAD or a NAD-like compound andthe agent to be tested, thereby producing a combination; detecting theNAD-dependent acetylation status of an amino acid in the histoneprotein; and comparing the NAD-dependent acetylation status in thepresence of the agent to be tested with the NAD-dependent acetylationstatus of the amino acid in the histone protein in the absence of theagent to be tested, wherein a difference in the NAD-dependentacetylation status of the amino acid of the histone protein between thepresence of the agent and the absence of the agent indicates that theagent alters the NAD-dependent acetylation status of at least one aminoacid of the histone protein.

The agent identified by the methods of the invention can add an acetylgroup or remove an acetyl group from at least one amino acid residue(e.g., lysine) of a histone protein (e.g., H2B, H3 or H4).

The invention further relates to a method of identifying an agent whichalters life span of a cell by altering the activity of a Sir2 protein ora Sir2-like protein by assessing the NAD-dependent acetylation status ofat least one amino acid (e.g., lysine) in a histone protein (e.g., H2B,H3 or H4). The histone protein, a Sir2 protein or Sir2-like protein, NADor a NAD-like compound and the agent to be tested are combined toproduce a combination, the NAD-dependent acetylation status of an aminoacid in the histone protein is detected (e.g., electron-spray or matrixassisted laser desorption/ionization (MALDI) mass spectroscopy); andcompared the NAD-dependent acetylation status in the presence of theagent to be tested with the acetylation status of the amino acid in thehistone protein in the absence of the agent to be tested. A differencein the acetylation status of the amino acid of the histone proteinbetween the presence and absence of the agent alters the life span ofthe cell. The agent tested can increase the lifespan of a cell byNAD-dependent deacetylation of the histone protein. Alternatively, oradditionally, the agent can decrease the lifespan of the cell byNAD-dependent acetylation of histone proteins. The agent can be anagonist of Sir2 activity or the agent can be an antagonist of Sir2activity.

The invention also provides a method of identifying an agent whichalters aging of a cell by altering the activity of Sir2 or a Sir-2 likeprotein by assessing the NAD-dependent acetylation status of at leastone amino acid (e.g., lysine) in a histone protein (e.g., H2B, H3 orH4). The histone protein, a Sir2 protein or Sir2-like protein, NAD or aNAD-like compound and the agent to be tested are combined to produce acombination, the NAD-dependent acetylation status of an amino acid inthe histone protein is detected (e.g., electron-spray massspectroscopy); and compared the NAD-dependent acetylation status in thepresence of the agent to be tested with the acetylation status of theamino acid in the histone protein in the absence of the agent to betested. A difference in the acetylation status of the amino acid of thehistone protein in the presence of the agent alters aging of the cell.The agent tested can increase the aging of a cell by NAD-dependentacetylation of the histone protein. Alternatively, the agent candecrease aging of the cell by NAD-dependent deacetylation of histoneproteins. The agent can be an agonist of Sir2 or an antagonist of Sir2.

Agents identified by the in vitro methods of the invention, such as themethod described above, can be further evaluated for their ability toalter the NAD-dependent acetylation status in a cell by administeringthe identified agents to a cell and monitoring the NAD-dependentacetylation status of at least one amino acid in a histone protein ofthe cell. It is expected that agents identified in vitro as agents whichalter the NAD-dependent acetylation status of a histone protein willalter the NAD-dependent alteration status of histone protein in vivo.Likewise, it is expected that the agents which alter NAD-dependentacetylation status of histone proteins by the methods described hereinwilt alter the lifespan and aging of a cell and/or organism.

The invention also relates to a method of altering the NAD-dependentacetylation status of at least one amino acid residue in a histoneprotein comprising combining the histone protein, a Sir2 protein orSir2-like protein and a NAD or a NAD-like compound. The method can beused in vitro (e.g., the absence of cells or an animal) or in vivo(e.g., in cells or animals).

The present invention also relates to a method of identifying agentswhich alter mono-ADP-ribosylation of one or more nuclear proteins in acell. The cell and agent to be tested are combined and the level ofmono-ADP-ribosylation of a nuclear protein, such as one or more histoneproteins (e.g., H2B, H2A, H3, H4), is determined and compared to thelevel of mono-ADP-ribosylation of that protein or proteins in theabsence of the agent. A difference in the level of mono-ADP-ribosylationin the presence and absence of the agent indicates that the agent altersthe mono-ADP-ribosylation of the nuclear protein. That is, if the levelof mono-ADP-ribosylation is greater in the presence of the agent than inthe absence of the agent, the agent is an agonist of mono-ADP-ribosylaseactivity. Similarly, if the level of mono-ADP-ribosylation is greater inthe absence of the agent than in the presence of the agent, the agent isan antagonist of mono-ADP-ribosylation.

Alternatively, the method of identifying agents which alterNAD-dependent acetylation status of a histone protein and/ormono-ADP-ribosylation of one or more nuclear proteins can be carried outat the organism (e.g. animal) level. An agent to be tested isadministered to an organism, and the alteration in NAD-dependentacetylation of a histone protein and/or the level ofmono-ADP-ribosylation of one or more nuclear proteins, such as one ormore histone proteins, in cells of the organism is determined andcompared to the acetylation status of a histone protein and/or the levelof mono-ADP-ribosylation of that protein or proteins in the absence ofthe agent. A difference in the acetylation status of a histone proteinand/or the level of mono-ADP-ribosylation in the presence and absence ofthe agent indicates that the agent alters NAD-dependent acetylationstatus of histone proteins and/or the mono-ADP-ribosylation of thenuclear protein.

Agents to be tested for activity in the assays described herein caninclude proteins (including post-translationally modified proteins),peptides (including chemically or enzymatically modified peptides), orsmall molecules (including carbohydrates, steroids, lipids, anions orcations, drugs, small organic molecules, oligonucleotides, antibodies,and genes encoding proteins of the agents or antisense molecules),including libraries of compounds. The agents can be naturally occurring(e.g., found in nature or isolated from nature) or can be non-naturallyoccurring (e.g., synthetic, chemically synthesized or man-made). Agentswhich alter the level of NAD-dependent acetylation status of histoneproteins or the mono-ADP-ribosylation of nuclear proteins of theinvention can be agonists (e.g., stimulators/enhancers) or antagonists(e.g., inhibitors) of NAD-dependent acetylation or mono-ADP-ribosylaseactivity. In a particular embodiment, the agents are agonists orantagonists of Sir2 (e.g., mSir2α, ySir2) dependent NAD-dependentacetylation and mono-ADP-ribosylation of histone proteins (e.g., H2A,H-2B, H3, H4).

The term “agonist” as used herein, refers to an agent which simulates ormimics NAD-dependent acetylation or deacetylation of histone proteins(e.g., mSir2, ySir2, human Sir2), a mono-ADP-ribosylase (e.g., mSir2α,ySir2, human Sir2) by, for example, deacetylating H3 or H4 (e.g.,removal an acetyl group from at least one lysine residue in the aminoterminus) and/or catalyzing the transfer of an adenosine diphosphateribose unit from an ADP donor (e.g., NAD) to an amino acid residue(e.g., lysine) of a substrate (e.g., histone protein H2B, H3, H4).Alternatively, or additionally, an agonist can be an agent whichstimulates, augments, enhances, increases, intensifies or strengthensNAD-dependent acetylation/deacetylation and/or mono-ADP-ribosylaseactivity or the interaction between a nuclear protein and aNAD-dependent acetylase, mono-ADP-ribosyltransferase, or alters amono-ADP-ribosylase in a manner (e.g., results in a conformationalchange) which augments the activity of the mono-ADP-ribosylase such thatthe nuclear protein is mono-ADP-ribosylated at a higher rate or ingreater quantities than in the absence of the agonist. An agonist canalso enhance the rate of removal of acetyl groups from amino acidresidues of histone proteins.

An agonist can also enhance the availability or accessability of amono-ADP-ribosylase to a substrate, or a substrate to amono-ADP-ribosylase, thereby further increasing the level ofmono-ADP-ribosylation of a nuclear protein. For example, a substancepossessing agonist activity can increase the rate at which ADP-ribose istransferred to an amino acid residue of a histone protein, such as H2B,H2A, H3, or H4 beyond that observed in the absence of the agonist.

The term “antagonist”, as used herein, includes a substance whichblocks, diminishes, inhibits, hinders, limits, decreases, reduces,restricts or interferes with the NAD-dependent acetylation/deacetylationof histone proteins and/or enzymatic activity of a mono-ADP-ribosylase,alters the substrate (e.g., a histone protein such as H2B, H2A, H3 orH4). An antagonist can act in a manner which prevents Sir2 fromdeacetylating an amino acid residue in a histone protein and/or amono-ADP-ribosylase (e.g., mSir2α, ySir2, human Sir2) from acting on thesubstrate, or interferes with the accessibility of the substrate (e.g.,H2B, H2 or H4) to Sir2 for alterations in NAD-dependent acetylationstatus and/or the mono-ADP-ribosyltransferase, or any combinationthereof.

Alternatively or additionally, an antagonist can prevent, impede orinterfere with the interaction between Sir2 and NAD, amono-ADP-ribosyltransferase and a nuclear protein substrate by alteringthe Sir2 or mono-ADP ribosyltransferase (e.g., conformationally),thereby preventing the Sir2 and/or a mono-ADP-ribosyltransferase fromaltering the NAD-dependent acetylation status of a histone proteinand/or catalyzing the transfer of an adenosine diphosphate ribose unitfrom an ADP donor (e.g., NAD) to an amino acid residue (e.g., threonine)of a substrate (e.g., a nuclear protein such as a histone protein). Forexample, a substance possessing antagonist activity can decrease therate at which the status of NAD-dependent acetylation (e.g.,deacetylation) takes place.

An agonist can also prevent, impede or interfere with ADP-ribosetransfer to an amino acid residue of a histone protein, such as H2B,H2A, H3 or H4, relative to that observed in the absence of theantagonist.

The agonist or antagonists of the present invention can also altertranscription of NAD-dependent acetylases and/ormono-ADP-ribosyltransferase genes (e.g., SIR2), the mRNA stability ofNAD-dependent acetylases and/or mono-ADP-ribosyltransferase transcripts,or degradation of the enzyme as a means to alter NAD-dependent acetylaseand/or mono-ADP-ribosyltransferase activity. For example, an agonist canincrease the level (e.g., total cellular pool) of deacetylated histoneproteins by increasing the activity of Sir2 in an NAD-dependent manner.Likewise agonists of the invention can also mono-ADP-ribosylated nuclearprotein by increasing transcription of mono-ADP-ribosyltransferaseenzyme genes (e.g., SIR2α) or decreasing enzyme degradation in a cell.

Similarly, an antagonist can decrease the levels of deacetylated histoneproteins or increase the amount of acetylated histone proteins.Antagonists can also decrease the levels of mono-ADP-ribosylation of anuclear protein by down regulating transcription ofmono-ADP-ribosyltransferase genes or increasing degradation of theenzyme.

As used herein, a “cell” refers to a single cell or a collection of atleast two or more cells. The cell can be a yeast cell (e.g.,Saccharomyces cerevisiae) or a mammalian cell, including but not limitedto, somatic or embryonic cells, Chinese hamster ovary cells, HeLa cells,human 293 cells and monkey COS-7 cells. The collection of cells can forma tissue or an organism. An “organism” refers to any living individual,such as a vertebrate animal (e.g., a mammal, including but not limitedto, mice, rats, pigs dogs, cats, primates, and humans) or nonvertebrateanimal, such as an insect (e.g., Drospholia melanogaster) or nematode(e.g., C. elegans), considered as a whole. A “tissue” refers to acollection of similar cell types (such as epithelium, connective, muscleand nerve tissue).

The term “mono-ADP-ribosylation”, as used herein, refers to the transferof one adenosine diphosphate ribose unit from an ADP donor (e.g., NAD)to an amino acid residue (e.g., threonine) of a substrate (e.g., one ormore histone proteins such as H2B). A “mono-ADP-ribosyltransferase”(also referred to herein as mono-ADP ribosylase is a protein (alsoreferred to herein as a polypeptide) which catalyzes themono-ADP-ribosylation. For example, mono-ADP-ribosyltransferasesencompassed by the invention include murine (m) SIR2 genes and proteins(e.g., mSir2α, mSir2β, mSir2γ), human Sir2 genes (Frye, Biochem.Biophys. Res. Commun. 260:273-279 (1999)) and proteins, yeast genes andproteins that are Homologous to SIR2 (HST) (e.g., HST1, HST2, HST3,HST4), and nucleic acid molecules having accession numbers AI466061,AI465820, or AI465098. (See FIG. 19). Database accession numbers for thenucleotide and amino acid sequences for some of thesemono-ADP-ribosyltransferases are known. (Frye, Biochem. Biophys. Res.Commun. 260:273-279 (1999)). A mSIR2β EST cDNA clone (SEQ ID NO: 34, 35)557657 can be purchased from Genome Systems, Inc.

Other known mono-ADP-ribosyltransferases include bacterial toxins, suchas cholera, pertussis and diphtheria toxins and mammalianglycosylphosphatidylinositol (GP1)-anchored mono-ADP-ribosyltransferases(Domenighini, et al., Microbiology 21:667-674 (1996); Moss, et al., Mol.Cell. Biochem. 193:109-113 (1999)). It is understood that any proteinthat mono-ADP-ribosylates nuclear proteins, in particular histoneproteins such as H2B, H2A, or H3, is within the scope of the invention.

A “level of mono-ADP-ribosylation” refers to an amount of a substrate towhich an adenosine diphosphate ribose has been transferred. The level ofmono-ADP-ribosylation can be assessed using methods known in the art,such as in vitro labeling techniques, for example, by monitoring theaddition of [³²P]NAD+ to a substrate (e.g., H2A, H2B, H3). By way ofillustration only, the ADP-ribosylase activity of an agent and the levelof mono-ADP-ribosylation of a substrate can be determined by adding aprotein to be evaluated (about 0-1 g) to a reaction buffer comprising 50mM Tris-HCl, pH 8.0, 4 nM MgCl₂, 0.2 mM DTT, 1 μM cold ornonradiolabeled NAD, 0.08 μM [³²P]NAD and admixing or gently vortexingto dilute, resuspend or mix the protein. Substrates (e.g., histoneproteins at a concentration of about 0-1 g) are then added and thereaction mixture is incubated at ambient temperature (18-25° C.) for30-120 minutes. The presence or absence of ADP-ribosylation products(e.g., ADP-ribosylated nuclear proteins) is detected usingautoradiography. Differences in the levels of mono-ADP-ribosylation, forexample in the presence and absence of an agent to be tested, can bedetermined using suitable techniques, including, but not limited to,densitometric scanning of autoradiographs or phosphoimaging techniquesof gels. (See, Ausubel, et al., “Current Protocols in Molecular Biology”John Wiley & Sons (1999)). Techniques to perform themono-ADP-ribosylation assays are detailed in the ExemplificationSection.

Confirmation of mono-ADP-ribosylation of substrates and, thus,mono-ADP-ribosyltransferase activity of an agent, can be performed, forexample, by adding a suitable amount of snake venom phosphodiesterase(e.g., 2 mg/ml, specific activity 1.5 U/mg) to the resulting product ofthe reaction mixture described above. The reaction product andphosphodiesterase are incubated at about 37° C. for about an hour.Absence of an autoradiographic band following phosphodiesterasedigestion, as compared with presence of an autoradiographic band in theabsence of digestion, indicates that the substrate wasmono-ADP-ribosylated (FIG. 4E). Mono-ADP-ribosylase activity can also beverified by the addition of one or more specific mono-ADP-ribosylationinhibitors, including, but not limited to, novobiocin and coumermycinA1, to in vitro assays described above (FIG. 4F). The inhibitor(s) canbe added before or after the addition of the substrate. The absence of aband in an autoradiograph following the addition of a specificmono-ADP-ribosylation inhibitor indicates that the agent hasmono-ADP-ribosylase activity. Thus, the mono-ADP-ribosyltransferaseactivity of agents is evaluated by mono-ADP-ribosylation of substrateproteins as described in the Exemplification Section.

In a preferred embodiment, agonists of the invention are agonists ofSir2 NAD-dependent acetylation of histone proteins and Sir2mono-ADP-ribosyltransferase activity. The identification of agonists orantagonists of Sir2 activity can be initially evaluated using yeastcells. In the presence of an agonist of Sir2 activity, a yeast cell willproduce a red colonies, whereas antagonists of Sir2 will result in awhite colony. When the ADE2 gene is inserted into one of the threeSir2-regulated silencing loci (e.g., telomere rDNA, or HM mating loci)it is silenced in the presence of the agonists, which eventually leadsto yeast cells accumulating red pigment. When the ADE2 gene istranscribed in the presence of antagonists, yeast cells metabolizing theintermediate red pigments and become white in color. Prescreening agentsemploying color selection of yeast cells will be particularly useful forlarge scale screening of agents which alter the activity ofNAD-acetylation of histone proteins and mono-ADP-ribosyltransferasesthat ADP-ribosylate nuclear proteins. Following a color selectionscreening protocol agents can be further evaluated for their ability toalter NAD-dependent acetylation of histone proteins and the level ofmono-ADP-ribosylation of nuclear proteins using techniques describedherein.

The mono-ADP-ribosyltransferases described herein catalyze the additionof ADP-ribose to cellular proteins, specifically nuclear proteins. A“nuclear protein” refers to any protein, polypeptide or peptide that islocated in or performs a function in the nucleus of a eukaryotic cell(e.g., a yeast cell, a zebrafish cell, a C. elegans cell, Drosophilamelanogaster cell, or a mammalian cell, such as a murine cell or a humancell). In a preferred embodiment, the nuclear proteins are histoneproteins. Histone proteins are highly conserved DNA-binding nuclearproteins that form the nucleosome, the basic subunit of the chromatin.Histone proteins can be one or more core histone proteins (e.g., H2A,H2B, H3, H4) or an outer histone protein (e.g., H1), or combinationsthereof. In a more preferred embodiment, the substrate formono-ADP-ribosylation is histone H2B or H3.

The term “alters” is defined as a change in an activity (e.g.,NAD-dependent acetylation status of at least one amino acid of a histoneprotein, mono-ADP-ribosylation) or phenomena (e.g., aging, life span),for example, relative to an activity or phenomena in the absence of anagent to be tested. Alteration includes both an increase and a decrease(e.g., complete abolishment), either qualitatively, quantitatively orboth, in the activity or phenomena being monitored.

The agent to be tested (e.g., an agonist or antagonist) can be combinedwith the cell before or after the addition of the substrate (e.g., ahistone protein such as H2B, H3, and/or H4). Experimental conditions forevaluating test substances, such as buffer (e.g., 50 mM Tris-HCl, pH8.0) or media, concentration (about 0-1 g), temperature (about 18-25°C.), and incubation (about 30-120 minutes) requirements, can, initially,be similar to those described in the Exemplification Section hereinrelating to NAD-dependent acetylation status of histone proteins andmono-ADP-ribosylation of histone proteins by mSir2α or ySir2. One ofordinary skill in the art can determine empirically how to varyexperimental conditions depending upon the biochemical nature of theagent and the particular cell used in the methods described herein.

If the agent to be tested is being administered to an organism, variousmethods and routes of administration are known in the art. The agentwill preferably be formulated in a pharmaceutical composition. Forinstance, suitable agents can be formulated with a physiologicallyacceptable medium to prepare a pharmaceutical composition. Theparticular physiological medium may include, but is not limited to,water, buffered saline, polyols (e.g., glycerol, propylene glycol,liquid polyethylene glycol) and dextrose solutions. The effective amountand optimum concentration of the active ingredient(s) (e.g., the agent)in the chosen medium can be determined empirically, according toprocedures well known to medicinal chemists, and will depend on theultimate pharmaceutical formulation desired. Methods of administrationof compositions for use in the invention include, but are not limitedto, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intraocular, oral and intranasal. Other suitable methodsof introduction can also include rechargeable or biodegradable devicesand slow release polymeric devices. The pharmaceutical compositions ofthis invention can also be administered as part of a combinatorialtherapy with other agents.

The invention further relates to the discovery that increasedNAD-dependent deacetylation of histone protein by Sir2 and/ormono-ADP-ribosylation of one or more nuclear proteins (e.g., histoneproteins) correlates with decreased rDNA recombination, increased lifespan and decreased aging at the cellular level. Thus, agents whichincrease NAD-dependent deacetylation of histone proteins by Sir2 and/ormono-ADP-ribosylation of nuclear proteins are expected to be agentswhich decrease rDNA recombination, increase life span and decreaseaging. Accordingly, in another embodiment, the methods of the inventionrelate to identifying agents which alter the life span of a cell or anorganism. It is envisioned that the methods of the invention can be usedfor any cell that undergoes aging, for example, a mammalian cell (e.g.,from a human), a zebrafish cell, a C. elegans cell, or Drosophilamelanogaster cell in, for example, experimental systems.

The cell or organism is combined with the agent to be tested and thelevel of NAD-dependent acetylation or deacetylation of histone proteinsby Sir2 and/or mono-ADP-ribosylation of one or more nuclear proteins isdetermined in the presence of the agent and compared to the level in anappropriate control in the absence of the agent. A difference in thelevel NAD-dependent acetylation of histone proteins and/or ofmono-ADP-ribosylation of the nuclear protein in the presence of theagent indicates that the agent alters NAD-dependent acetylation ofhistone proteins and/or the mono-ADP-ribosylation of a nuclear proteinof a cell or an organism and that the agent alters life span of the cellor organism.

Methods of determining life span of a cell or an organism are known inthe art. With regard to a yeast cell, for example, “life span” refers tothe number of generations, or divisions of a mother cell, which giverise to daughter cells. Techniques to assess the life span of yeastcells, including growth conditions for particular strains, are wellknown and include stress-resistance methods, cell surface labelingmethods and temperature sensitive methods. (See, for example, Guarenteet al., U.S. Pat. No. 5,874,210 (1999), the teachings of which areincorporated herein in their entirety). The life span of a yeast cell inthe presence of an agent can be initially identified, e.g., by the meannumber of generations using a colony color selection strategy. Theability of an agent to alter (e.g., increase) the life span of a cell,as described herein, is measured as an increase in the mean life span ofa cell(s) as compared with the mean life span of a cell(s) not combinedwith the test agent. For example, the life span of the yeastSaccharomyces cerevisiae can be increased at least 2-3 fold in thepresence of two copies of the yeast or mammalian (e.g., murine, human)NAD-dependent deacetylase and/or mono-ADP-ribosyltransferase SIR2 genecompared to cells lacking the SIR2 gene (FIG. 20). When SIR2 is deletedfrom yeast haploid cells the life span of cells is reduced by about 50%compared to wild type. When an extra copy of the SIR2 gene is introducedinto wild type yeast haploid cells (the yeast cells have two copies ofthe gene), the cells display a longer life span than wild type (Kennedy,et al., Cell 90:485-496, (1995); Kaeberlein, et al., Genes &Development, 13:2370-2580, (1999)).

The life span of mammalian cells (e.g., human and murine diploidfibroblasts, epithelial cells and lymphocytes) refers to the number ofpopulation doublings until the cells are in a growth-arrested state suchas “cellular senescence” (Hayflick et al., Experimental Cell Research,25:585-621, (1961); Todaro, et al., Journal of Cell Biology, 17:299-313, (1963); Rohme, Proc. Natl. Acad. Sci. USA, 78:5009-5013,(1981)). For some mammalian cells which obtain the ability toproliferate indefinitely in culture (e.g., cell lines established fromcancers and cell lines engineered chemically or genetically to haveinfinite life span), the life span can be artificially terminated by themethods of the invention. In this case, the life span is defined as theaverage doubling cycles before the cells die by the induction ofcellular senescence or apoptosis.

The life span of an organism (e.g, vertebrate organism, such as a human,or a nonvertebrate organism, such as an insect, nematode or fish) refersto the average (also referred to herein as mean) duration of life of agiven organism. For example, the average life span of a male human beingis about 71.8 years, and the average life span of a female human beingis about 78.6 years and the average life span of a mouse is about0.7-2.7 years (Finch, “Longevity, Senescence and the Genoma,” Univ. ofChicago Press, Chicago, Ill. (1990)).

In one embodiment, the agent increases the life span of the cell ororganism by increasing the NAD-dependent deacetylation of histoneproteins, in particular at least one amino acid at the N-terminal lysineresidues of H3 (e.g., lysine 9 and/or lysine 14) and/or H4 (e.g., lysine16), by increasing the activity of Sir2. In another embodiment, theagent increases the lifespan of the cell or organism by increasing thelevel of mono-ADP-ribosylation of a nuclear protein, such as H2B or H3,in particular mono-ADP-ribosylation of a lysine residues in H3 and/orH4. An increase in life span would be a duration of a particular lifebeyond the average for that cell or organism.

In another embodiment, the agent decreases the life span of the cell ororganism by decreasing the activity of Sir2, thereby alteringNAD-dependent acetylation (e.g., deacetylation) of histone proteins, inparticular at least one amino acid at the N-terminus of H3 (e.g., lysine9 and/or lysine 14) and/or H4 (e.g., lysine 16). In an additionalembodiment, the agent decreases the level of mono-ADP-ribosylation of anuclear protein, such as H2B or H3. A decrease in life span would aduration of a particular life less than average for that cell organism.

The invention also pertains to a method of identifying an agent whichalters aging of a cell or an organism. The cell or organism and agent tobe tested are combined and the level of NAD-dependent acetylation ofhistone proteins and/or the level of mono-ADP-ribosylation of a nuclearprotein (e.g., H2B, H2A or H3) is determined and compared to theNAD-dependent acetylation status of a histone protein and/or the levelof mono-ADP-ribosylation of the nuclear protein in the absence of theagent. A difference in the NAD-dependent acetylation status and/or levelof mono-ADP-ribosylation in the presence and absence of an agentindicates that the agent alters aging of the cell or organism.

“Aging” refers to all time-dependent changes to which biologicalentities, from molecules to ecosystems, are subject, though themechanisms and consequences to function may be vastly different(Medawar, “An Unsolved Problem of Biology”, H. K. Lewis, London, (1952).Additionally or alternatively, aging refers to the process of growingold or senencing. Aging is associated with particular phenotypes in acell or organism. In a cell, aging can be characterized by an increasein cell size, a slowing of the cell cycle, shortening of telomeres,and/or expression of some particular categories of genes(Stanulis-Praeger, Mech. Ageing Dev. 38:1-48, (1987); Faraher, et al.,BioEssay, 20:985-991, (1998)). The changes in heterochromatin structureand mitochondria can also be associated with aging (Imai, et al.,Experimental Gerontology, 33:555-570, (1998); Wallace, et al.,Biofactors, 7:187-190, (1998)). In yeast cells, sterility, theappearance of surface wrinkles, blebs and bud scars, nucleolarenlargement and fragmentation, and formation, replication, andaccumulation of rDNA circles are also associated with aging (Sinclair,Mills and Guarente, Annu. Rev. Microbiol, 52533-560, (1998)).

In yeast, aging is defined by the relatively fixed number of celldivisions undergone by mother cells (Müller et al., Aging Dev. 12:47(1980)), as well as characteristic changes during their life span(Jazwinski, Science 273:54 (1996)), such as cell enlargement (Mortimerand Johnson, Nature 183:1751 (1959)) and sterility (Müller, J.Microbiol. Serol. 51:1 (1985)). Techniques to determine aging in a yeastcell are well-known and are described, for example, in Guarente et al.,U.S. Pat. No. 5,874,210 (1999), Smeal, et al., Cell 84:633-642 (1996);Sinclair, et al., Science 277:1313-1316 (1997); Sinclair, et al., Cell91:1033-1042 (1997)), the teachings of all of which are incorporatedherein in their entirety.

Aging of an organism refers to the deterioration of the organismassociated with changes in structures and functions (e.g., phenotype)that are characteristic of a particular organism. Such characteristicscan include, for example, hair loss, graying of hair, osteoporosis,cataracts, atherosclerosis, loss of skin elasticity and a propensity forcertain cancers.

The agents identified by the methods of the present invention can beused to alter the aging of a cell or an organism by altering the levelSir2 activity, NAD-dependent acetylation status of histone proteinsand/or of mono-ADP-ribosylation of a nuclear protein. In a preferredembodiment, the agents identified by the methods of the inventiondecrease aging of the cell or organism. In another embodiment, theagents identified by the methods of the invention increase aging of thecell or organism.

Due to their ease of experimental manipulation, similar structuralfeatures associated with aging in mammalian cells (Guarente et al., U.S.Pat. No. 5,874,210 (1999)), and an Sir2 protein which shares sequence(nucleic acid and amino acid) and enzymatic (e.g., NAD-dependentacetylation of histone proteins, mono-ADP-ribosyltransferase) with amammalian Sir2 proteins (e.g., mSir2α, mSir2β), the yeast cell is asuitable cellular model to identify agents which alter NAD-dependentacetylation of histone proteins or mono-ADP-ribosylation of nuclearproteins, increase life span and decrease aging in mammalian cells andorganisms as well as nonmammalian cells and organisms (e.g., insect,nematode, fish). Moreover, a mammalian (e.g., mSir2α) NAD-dependentdeacetylase and/or mono-ADP-ribosylase increases the life span and slowsaging of yeast cells. Thus, mammalian and yeast Sir2 proteins mediatesimilar biological activities.

As defined herein, mammals include rodents (such as rats, mice or guineapigs), domesticated animals (such as dogs or cats), ruminant animals(such as horses, cows) and primates (such as monkeys or humans).

Thus, the agents identified by the methods described herein, whichincrease life span or decrease aging as described above, can be used toincrease the life span or decrease aging of cells, e.g., mammalian cellsincluding human cells, or vertebrate (e.g., mice, humans, guinea pigs,rats) or invertebrate (e.g., insects, nematodes) organisms. For example,cells to be treated can be combined with an effective amount of an agentthat increases the activity of Sir2, increases the NAD-dependentdeacetylation of histone proteins, and/or level of mono-ADP-ribosylationof a nuclear protein, thereby increasing their life span or decreasingtheir aging relative to untreated cells or organisms.

An “effective amount” of an agent, as used herein, is defined as thatquantity of an agent which increases life span or decreases aging, toany degree, in the cell or organism being treated by increasing, to anydegree, the NAD-dependent deacetylation of histone proteins by Sir2 or aSir2-like protein and/or the level of mono-ADP-ribosylation of a nuclearprotein (e.g., H2B, H2A, H3, or H4). In a preferred embodiment, lysinesat position 9 and/or 14 in H3 are deacetylated and/or lysine at position16 in H4 is deacetylated. For example, an effective amount of an agonistof a mono-ADP-ribosyltransferase in a cell can inhibit certaincharacteristics associated with aging, for example, the formation ofrDNA with, or without, inhibiting other characteristics, such as thereplication and/or accumulation of rDNA. Similarly, an effective amountof an agent that increases the NAD-dependent deacetylation of histoneproteins (e.g., H2B, H3 and/or H4) and/or the levels ofmono-ADP-ribosylation of a nuclear protein can decrease aging in anorganism by slowing hair loss, with or without, diminishing graying orhair, cataracts or atherosclerosis.

It is envisioned that the agents identified by the methods of thepresent invention can be administered to cells or organisms which agenaturally (e.g., normally) or age prematurely or abnormally (e.g., as aresult of Werner's syndrome in mice or humans) due to geneticpermutations. Thus, the agents identified by the methods of the presentinvention, can be useful in anti-aging therapies and for the treatmentof aging diseases, such as Werner's syndrome, to prevent, halt, hasten,slow or ameliorate premature aging. Additionally, or alternatively, theidentification of agents which alter the NAD-dependent acetylationstatus of histone proteins (e.g., lysines 9 and/or 14 of H3, lysine 16of H4) and/or the mono-ADP-ribosylation of nuclear proteins can be alsobe useful for the continued study of cellular and organismal agingprocesses.

Thus, the present invention also pertains to methods of increasing thelife span or decreasing the aging of a cell or an organism byadministering an effective amount of an agent that increases theNAD-dependent deacetylation of histone proteins and/or increases themono-ADP-ribosylation of a nuclear protein. The agent is identified bycombining an agent to be tested with a cell or an organism anddetermining the level of NAD-dependent deacetylation of histone proteins(e.g., H3, H4) and/or mono-ADP-ribosylation of a nuclear protein in thepresence of the agent which is compared to the NAD-dependent acetylationof a histone protein and/or level of mono-ADP-ribosylation of a nuclearprotein in the absence of the agent. The presence of an agent whichincreases life span or decreases aging increases the activity of Sir2thereby increasing the deacetylation of histone proteins and/orincreases the level mono-ADP-ribosylated nuclear protein (e.g., H2B,H2A, H3, H4).

The present invention also pertains to methods of increasing the lifespan or decreasing aging of a cell or an organism by administering tothe cell or organism an agonist of Sir2 that increases the NAD-dependentdeacetylation of histone proteins, a mono-ADP-ribosyltransferase (e.g.,Sir2) or an agonist of a mono-ADP-ribosyltransferase of a nuclearprotein. In a preferred embodiment, the nuclear protein a histoneprotein, in particular H2A, H2B, H3 or H4. In a more preferredembodiment, the acetylation status of lysine residues at positions 9and/or 14 of H3 are altered and the lysine at position 16 of H4 isaltered by altering the activity of Sir2. Preferably themono-ADP-ribosyltransferase is a Sir2 protein (e.g., Sir2α, Sir2β,Sir2γ). Specifically, the core domain of a Sir2 protein (FIG. 2B, 6A, 14a, 19; SEQ ID NOS: 2, 3, 4, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23 and 24) alters the NAD-dependent acetylation statusof histone proteins, the mono-ADP-ribosyltransferase or the agonist ofNAD-dependent acetylation status of histone proteins and/ormono-ADP-ribosyltransferase activity of the core domain of Sir2.

Because aging in cells is associated with the formation, replicationand/or accumulation of ribosomal DNA (rDNA) circles, the presentinvention relates to methods of inhibiting the formation, replicationand/or accumulation of rDNA circles in a cell or an organism byadministering an effective amount of a mono-ADP-ribosyltransferase oragonist of NAD-dependent acetylation status of histone proteins and/or amono-ADP-ribosyltransferase to a cell or organism. rDNA circlesaccumulate exponentially in old yeast mother cells and are responsiblefor age-related enlargement and fragmentation of the nucleolus, andappear to nucleate the fragmented nucleolus. (Sinclair, et al., Cell91:1033-1042 (1997)). For example, agents which have been identified bymethods described herein as agonists of NAD-dependent deacetylationand/or of mono-ADP-ribosylation of nuclear proteins can be administeredto a cell or an organism to suppress the formation, replication and/oraccumulation of rDNA circles. The cell samples thus treated are thenexamined for longer-lived, slower aged colonies, using any of themethods described herein or other appropriate methods.

The invention further relates to a method of suppressing recombinationbetween rDNA in a cell or an organism comprising administering to thecell or the organism a mono-ADP-ribosyltransferase or an agonist ofNAD-dependent deacetylation of histone proteins and/or amono-ADP-ribosyltransferase. Recombination between rDNA is acharacteristic of aging in cells. In yeast cells, sterility associatedaging is due to a loss of transcriptional silencing at HMRa and HMLαloci, and the resulting expression of both a and α mating-typeinformation (Smeal et al., Cell 84:633 (1996)). The Sir2 proteinsilences genes inserted at rDNA (Bryk et al., Genes Dev. 11:255 (1997);Smith and Boeke, Genes Dev. 11:241 (1997)) and suppresses recombinationbetween rDNA repeats (Gottleib and Esposito, Cell 56:771 (1989)). Thus,suppressing recombination between rDNA by administering amono-ADP-ribosyltransferase or an agonist of a NAD-dependentdeacetylation of histone proteins and/or mono-ADP-ribosyltransferase canalso decrease aging and increase life span.

Agents which alter the NAD-dependent acetylation status of histoneproteins and/or mono-ADP-ribosylation of nuclear proteins (e.g., agonistof NAD-dependent deacetylation and/or mono-ADP-ribosyltransferaseactivity) can be formulated into compositions. Such compositions canalso comprise a pharmaceutically acceptable carrier, and are referred toherein as pharmaceutical compositions. The agonist or antagonistcompositions of the present invention can be administered intravenously,parenterally, orally, nasally, by inhalation, by implant, by injection,or by suppository. The agonist or antagonist compositions can beadministered in a single dose or in more than one dose over a period oftime to achieve a level of NAD-dependent deacetylation of histoneproteins and/or mono-ADP-ribosylation of nuclear protein which issufficient to confer the desired effect.

Suitable pharmaceutical carriers include, but are not limited to sterilewater, salt solutions (such as Ringer's solution), alcohols,polyethylene glycols, gelatin, carbohydrates such as lactose, amylose orstarch, magnesium stearate, talc, silicic acid, viscous paraffin, fattyacid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc. Thepharmaceutical preparations can be sterilized and if desired, mixed withauxiliary substances, e.g., lubricants, preservatives, stabilizers,wetting substances, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like which do notdeleteriously react with the agonist or antagonist compounds. They canalso be combined where desired with other active substances, e.g.,enzyme inhibitors, to reduce metabolic degradation.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Ampulesare convenient unit dosages.

It will be appreciated that the actual effective amounts of an agonistor antagonist of NAD-dependent acetylation status of histone proteinsand/or mono-ADP-ribosylation of nuclear proteins in a specific case canvary according to the specific agonist or antagonist being utilized, theparticular composition formulated, the mode of administration and theage, weight and condition of the patient, for example. For example, aneffective amount of an agonist is an amount of agonist capable ofslowing aging or extending the life span of a cell or an organism byaltering the NAD-dependent acetylation status and/ormono-ADP-ribosylating substrates such as nuclear proteins (e.g., histoneproteins, preferably H2A, H2B, H3). Dosages of suitable agonists orantagonists of NAD-dependent deacetylation of histone proteins and/ormono-ADP-ribosylation of nuclear proteins for a particular patient canbe determined by one of ordinary skill in the art using conventionalconsiderations, (e.g. by means of an appropriate, conventionalpharmacological protocol).

The present invention further relates to novel murine SIR2 (SilentInformation Regulator) genes (e.g., mSIR2α, mSIR2β) and their proteinproducts. The invention also relates to the discovery that mSir2α andyeast Sir2 (ySir2) proteins possess mono-ADP-ribosyltransferaseactivity, in particular mSir2α and ySir2 mono-ADP-ribosylate histoneproteins (e.g., H2B, H3, H4). As shown in the Exemplification Section,transfection of yeast cells, which lack SIR2, with ySIR2 genes, slowsaging and extends the life span of cell. Specifically, theadministration of two copies of the ySIR2 gene increases life expectancyof the cell about 1.5 fold over cells with a single copy of the SIR2gene and about 2-3 fold above cells which do not contain the SIR2 gene.Thus, the SIR2 gene of the present invention plays a critical role inthe life span and aging of a cell and, thus, of a multicellular organismsuch as a mammal.

The terms “SIR2” and “Sir2 amino acid sequence or Sir2 protein” refer tothe SIR2 gene and its protein product, respectively. SIR2 or Sir2 refersto the any of the yeast (e.g., S. cerevisiae) SIR2 or Sir 2. “mSIR2” or“mSir2” refers to any murine SIR2 (e.g., SIR2α, SIR2β, SIR2γ). The terms“SIR2 nucleic acid”, “nucleotide sequence of SIR2” or the “correspondingSIR2 nucleic acid”, refer to the nucleic acid sequence that encodes theSir2 protein.

The present invention also relates to the nucleic acid sequencesencoding murine Sir2 proteins (e.g., mSir2α, mSir2β, mSir2γ). Inparticular a full length cDNA and genomic DNA nucleic acid sequence ofmSIR2α and a partial cDNA sequence of SIR2β. The murine Sir2 proteins(e.g., mSir2α, mSir2β, mSir2γ) are encoded by at least three differentgenes. The nucleic acid sequence (SEQ ID NO.: 25) that encodes mSir2αprotein (SEQ ID NO.: 26) can be found in FIG. 21.

The Sir2 proteins have functional domains. For example, one domain canbe the core domain and another domain can be the amino-terminus orcarboxy-terminus. The core-domain of the Sir2 protein (e.g., mSir2α) canalter the NAD-dependent acetylation status of histone proteins and/ormono-ADP-ribosyltransferase activity. The invention is also intended toembody the functional domains (e.g., core-domain of Sir2), as describedherein. The amino acid sequence of the core domain of the mSir2α protein(SEQ ID NOS.: 4, 9, 12 and 19) is depicted in FIGS. 2C, 6, 14 a and 19.Amino acid sequences (SEQ ID NOS: 2, 3, 5, 10, 11, 13, 14, 15, 16, 17,18, 20, 21, 22, 23 and 24) which share structural identity to thenucleic acid and amino acid sequences of mSir2α are depicted in FIGS.2C, 6A, 14 a and 19.

The term “isolated”, as it refers to a Sir2 protein, means a protein, asfound in nature, but separated away from other proteins and cellularmaterial of their source of origin. The isolated Sir2 proteins includeessentially pure protein from cells, proteins produced by chemicalsynthesis, by combinations of biological and chemical synthesis and/orby recombinant methods.

The invention is intended to encompass Sir2 proteins, and proteins andpolypeptides having amino acid sequences analogous (also referred toherein as similar or homologous) to the amino acid sequences of the Sir2protein (e.g., mSir2α, ySir2) and functional equivalents or fragmentsthereof. Such proteins are defined herein as Sir2-like proteins, Sir2homologs (e.g., analogues) or derivatives. Any protein possessing theSir2 consensus sequence GAGISTS(L/A)GIPDFR (SEQ ID NO: 27) or YTQNID(SEQ ID NO: 28) (Brachmann, et al., Genes & Development 9:2888-2902(1995) and which is capable of NAD-dependent deacetylation and/ormono-ADP-ribosylation of nuclear proteins is within the scope of theinvention.

It is also envisioned that other Sir proteins (e.g., Sir1, Sir3, Sir4)or Sir-like proteins and the nucleic acids (e.g., SIR1, SIR3, SIR4)which encode Sir proteins, other than Sir2 or Sir2-like proteins, arewithin the scope of the invention.

Analogous or homologous amino acid sequences are further defined hereinto mean amino acid sequences with sufficient identity to the Sir2 aminoacid sequences so as to possess the biological activity of a Sir2protein (e.g., mono-ADP-ribosylation of histone proteins). For example,an analogous peptide can be produced with “silent” changes in amino acidsequence wherein one, or more, amino acid residues differ from the aminoacid residues of the Sir2 protein, yet still possess the biologicalactivity of Sir2. Examples of such differences include additions,deletions or substitutions of residues of the amino acid sequence ofSir2. Also encompassed by the invention are analogous proteins thatexhibit greater, or lesser, NAD-dependent deacetylation of histoneproteins and/or mono-ADP-ribosyltransferase activity (e.g., biologicalactivity) of the Sir2 protein of the present invention. Suchstructurally and functionally related proteins are also referred toherein as “Sir2-like” proteins.

The “biological activity” of a Sir2 protein is defined herein to meanthe ability to alter the NAD-dependent acetylation status of a histoneprotein and/or the mono-ADP-ribosylates a substrate such as a histoneprotein (e.g., H2B, H2A, H3, H4). In particular, the biological activityof mSir2α is the ability to alter the NAD-dependent acetylation statusof lysine residues in the N-terminus of H3 (e.g., lysine 9 and/or 14)and/or H4 (e.g., lysine 16) and/or mono-ADP-ribosylate an amino acidresidue of H2B, H3 or H4. Additionally, or alternatively, the biologicalactivity also means the ability to slow aging or extend life span by,for example, repressing recombination of rDNA, or inhibiting theformation, replication and/or accumulation of rDNA circles of a cell oran organism. The biological function of Sir2 is illustrated and furtherdefined by the Exemplification Section.

The invention also encompasses biologically active polypeptide fragmentsof the Sir2 protein, as described herein. Such fragments can includeonly a part of a full length amino acid sequence of Sir2 and yet possessthe ability to NAD-dependent deacetylation and/or mono-ADP-ribosylate asubstrate. Specifically encompassed by the invention are fragments ofSir2 comprising the core domain of Sir2.

The term “core domain” (also referred to herein as “core”) refers to theevolutionarily conserved domains of Sir2 or Sir2-like proteins which canbe identified, for example, by the comparison of amino acid sequencesby, for example, CLUSTAL X, BLAST, PSI-BLAST or FASTA algorithms. The“core domain” is the domain that shows significant identity and/orhomology to about 240-270 amino acids of Sir2 or Sir2-like proteins(about 20-50% or higher as amino acid identity, see FIG. 2) and/orpossesses the consensus sequence GAG(V/I)S(T/V)S(L/C/A)GIPDFRS (SEQ IDNO: 38) and YTQNID (SEQ ID NO: 28) (Brachmann, et al., Genes &Development 9:2888-2902, (1995)). The “core domain” of Sir2 proteins hasNAD-dependent deacetylation and/or mono-ADP-ribosylation activities. Anyprotein with a “core domain” of a Sir2 protein, a fragment of the coredomain, or any functional or structural equivalent which is capable ofNAD-dependent deacetylation and/or mono-ADP-ribosylation of nuclearproteins is within the scope of the invention.

The Sir2 protein and nucleic acid sequence include homologues, asdefined herein. The homologous proteins and nucleic acid sequences canbe determined using methods known to those of skill in the art. Initialhomology searches can be performed at NCBI against the GENBANK® (release87.0), EMBL (release 39.0), and SWISSPROT (release 30.0) databases usingthe BLAST and PSI-BLAST network services. (See, for example, Altshul, etal., J. Mol. Biol. 215: 403 (1990); Altschul et al. Nucleic Acid Res.25: 3389-3402 (1997); Altschul, Nucleic Acids Res. 25:3389-3402 (1998),the teachings of which are incorporated herein by reference). Computeranalysis of nucleotide sequences can be performed using the MOTIFS andthe FindPatterns subroutines of the GENETICS COMPUTING GROUP (GCG®,version 8.0) software. Protein and/or nucleotide comparisons can also beperformed according to CLUSTAL algorithms (Higgins, et al., Gene, 73:237-244 (1988)). Homologous proteins and/or nucleic acid sequences tothe Sir2 protein and/or nucleic acid sequences that encode the Sir2protein are defined as those molecules with greater than 25% sequencesidentity, also referred to herein as similarity, (e.g., 30%, 40%, 50%,60%, 70%, 80% or 90% homology). The percent identity between the yeast(S. cerevisiae) SIR2 sequence and the murine SIR2α and SIR2β sequence is45.9% (FIG. 2D). These particular sequence similarities and identitieswere determined using the CLUSTAL algorithm.

Homology was determined in accordance with the methods described inabove (e.g., BLAST, PSI-BLAST algorithms). Homologs of mSIR2α can be,for example, mSIRγ, mSIR2β, ySIR2, yHST1, yHST2, yHST3, yHST4 (aminoacid sequences depicted in SEQ ID NOS: 2, 3, 5, 10, 11, 13, 14, 15, 16,17, 18, 20, 21, 22, 23 and 24; FIGS. 2C, 6A, 14 a and 19) and human SIR2nucleic acid or protein (Frye, Biochem. Biophys. Res. Commun.260:273-279 (1999)).

Sequence identity of homologs of SIR2 can be to the entire nucleic acidor amino acid sequence, or to the core domain of the protein. In oneembodiment, the BLAST or PSI-BLAST parameters are set such that theyyield a sequence having at least about 60% sequence identity with thecorresponding known mSIR2α sequence, preferably, at least about 70%sequence. In another embodiment, the percent sequence identity is atleast about 85%, and in yet another embodiment, at least about 95%. Suchmolecules are also referred to herein as Sir2-like proteins. Thus, theSir2-like proteins possess structural identity with the SIR2 molecules(e.g., nucleic acid or amino acid identity) described herein. TheSir2-like proteins are capable of altering the NAD-dependent acetylationstatus of at least one amino acid in a histone protein (e.g. lysine 9and/or 14 of H3, lysine 16 of H4) and/or mono-ADP-ribosylatingsubstrates (e.g., histone proteins), thus extending the life span orslowing the aging of a cell or an organism.

Biologically active derivatives or analogs of the Sir2 protein can bemade using peptide mimetics, designed and produced by techniques knownto those of skill in the art. (see e.g., U.S. Pat. Nos. 4,612,132;5,643,873 and 5,654,276, the teachings of which are incorporated hereinby reference). These peptide mimetics can be based, for example, on aspecific Sir2 amino acid sequence such as the core domain of Sir2α.These peptide mimetics can possess biological activity similar to thebiological activity of the corresponding peptide compound, but possess a“biological advantage” over the corresponding Sir2 amino acid sequencewith respect to one, or more, of the following properties: solubility,stability and susceptibility to hydrolysis and proteolysis.

It is also envisioned that any NAD-dependent deacetylase that alters theacetylation status of at least one amino acid of a histone proteinand/or a mono-ADP-ribosyltransferase or Sir2-like protein containingproteins functionally equivalent to the Sir2 proteins described hereinwill be within the scope of the invention. The phrase “functionallyequivalent” as used herein refers to any molecule (e.g., peptide,peptide mimetic, protein and nucleic acid sequence encoding the protein)which mimics the NAD-dependent deacetylation activity of Sir2 and/ormono-ADP-ribosyltransferase activity of the Sir2α proteins and/orfunctional domains of Sir2 (e.g., the carboxy-terminus) described herein(such as Sir2β, Sir2γ, HST1, HST2, HST3) or which exhibit nucleotide oramino acid sequence identity to Sir2α such as Sir2β, Sir2γ, for example.

The Sir2 protein or Sir2-like protein can also be mutated todown-regulate or affect one or more properties of the protein, whileother properties still exist. In particular, the Sir2 protein can bemutated at a position in the sequence that prevents it from catalyzingthe transfer of an ADP ribose from a donor, such as NAD, to a substrateand/or from acetylating or deacetylating at least one amino acid of ahistone protein (e.g., lysine 9 and/or 14 of H3, lysine 16 of H4).

Thus, another embodiment of the present invention encompasses mutants ofSir2 proteins with altered NAD-dependent deacetylation activity and/ormono-ADP-ribosyltransferase activity. Mutants with NAD-dependentdeacetylation activity and/or mono-ADP-ribosyltransferase activitygreater than that of wildtype (e.g., nonmutant, naturally occurring)Sir2 are referred to as having “increased” NAD-dependent deacetylationactivity or “increased” mono-ADP-ribosyltransferase activity. Mutantswith a mono-ADP-ribosyltransferase activity less than that of wildtypeSir2 are referred to as having “decreased” NAD-dependent deacetylationactivity or “decreased” mono-ADP-ribosyltransferase activity. Themutants of the present invention can be used, for example, to furtherunderstand the mechanism of substrate specificity and cellular pathwaysinfluenced by NAD-dependent deacetylation of histone proteins and/ormono-ADP-ribosylation of substrates, such as histone proteins.

The term “mutant”, as used herein, refers to any modified nucleic acidsequence encoding a Sir2 protein or Sir2-like protein. For example, themutant can be a protein produced as a result of a point mutation or theaddition, deletion, insertion and/or substitution of one or morenucleotides encoding the Sir2 protein, or any combination thereof.Modifications can be, for example, conserved or non-conserved, naturalor unnatural.

As used herein a mutant also refers to the protein encoded by themutated SIR2 nucleic acid. That is, the term “mutant” also refers to aprotein (also referred to herein as polypeptide) which is modified atone, or more, amino acid residues from the wildtype (e.g., naturallyoccurring) Sir2 protein. In a preferred embodiment, mutants aregenerated by mutations in mSir2α or ySir2 proteins. In a particularembodiment, the core domain of the mSir2α protein, as described herein,has a mutation resulting in a altered mono-ADP-ribosyltransferaseactivity. For example, in this embodiment the mSir2α mutant is a mutantof mSir2α resulting from a point mutation substituting the glycine atposition 253 in the core domain of mSir2α with alanine residue togenerate the G253A Sir2α mutant. Sir2 mutants can be made by mutationsto one, or more, amino acid residues selected from a group consisting ofglycine, serine, proline, isoleucine, phenylalanine, threonine, orhistidine, or any combination thereof.

As used herein the designation for the amino acid substitutions for theSir2 mutants is indicated as, for example, “G253A”, wherein the letterto the left of the number indicates the amino acid in the wildtype Sir2protein (e.g., “G” or glycine); the number indicates the position of theamino acid in the wildtype Sir2 protein (e.g., position 253); and theletter to the right of the number indicates the amino acid residue whichreplaces the wildtype (e.g., “A” or alanine).

Additionally, or alternatively, the mutants are designated by the aminoacid which is mutated to an alanine residue, for example Thr-261,Gly-270, Iso-271, Arg-275 or Asn-345. Specifically encompassed by theinvention are mSir2α mutants G253A, G255A, S257A, I262A, F265A, R266A,G270A (also referred to herein as Gly-270), P285A, T336A, H355A; andySir2 mutants Thr-261, Iso-271, Arg-275, Asn-345 and Asp-347.

Using well-known techniques to align amino acids, amino acid residuessuitable for mutation as described herein for mSir2α or ySir2 proteinscan be determined for other Sir2 (e.g., Sir2β, Sir2γ) or Sir2-like(e.g., HST1, HST2) proteins. Nucleic acid sequences encoding the Sir2proteins can be mutated; the mutated nucleic acid constructs expressedunder standard experimental conditions well known to the skilledartisan; and the resulting mutant proteins evaluated for NAD-dependentdeacetylation of histone proteins and/or mono-ADP-ribosyltransferaseactivity, the ability to slow aging, or extend life span as describedherein.

It is also envisioned that fragments of the Sir2 proteins can be used inthe methods of the invention. “Fragments” of Sir2 proteins, as usedherein, refer to any part of the Sir2 protein capable of altering theNAD-dependent acetylation status of at least one amino acid residue of ahistone protein (e.g., lysine 9 and/or 14 of H3, lysine 16 of H4) and/ormono-ADP-ribosylating a substrate (e.g., H2B, H3, or H4). For example,the core domain of the Sir2 protein is considered a fragment of Sir2.

A Sir2 protein can be in the form of a conjugate or a fusion proteinwhich can be manufactured by known methods. Fusion proteins can bemanufactured according to known methods of recombinant DNA technology.For example, fusion proteins can be expressed from a nucleic acidmolecule comprising sequences which code for a biologically activeportion of the Sir2 protein and a fusion partner, for example, a portionof an immunoglobulin or a peptide linker. A nucleic acid constructencoding a Sir2 fusion protein can be introduced into a host cell,expressed, isolated or purified from a cell by means of an affinitymatrix employing antibodies to Sir2 or the fusion partner (e.g., IgG).

Antibodies can be raised to the Sir2 protein, Sir2-like protein,analogs, and portions thereof, using techniques known to those of skillin the art. (See, for example, Kohler et al., Nature 256:495-497 (1975);Cole et al., “Monoclonal Antibodies and Cancer Therapy”, Alan R. Liss,Inc., pp. 77-96 (1985); Ausubel et al., “Current Protocols in MolecularBiology”, John Wiley & Sons, Inc. (1999)). A mammal, such as a murine,rat, hamster or rabbit, can be immunized with an immunogenic form of theprotein (e.g., mSir2α, mSirβ, or a peptide comprising an antigenicfragment of the protein which is capable of eliciting an antibodyresponse). Techniques for conferring immunogenicity on a protein orpeptide include conjugation to carriers, such as BSA or keyhole limpethemocyanin, or other techniques, such as the use of Freud's adjuvant orTITERMAX®, well known in the art. The progress of immunization can bemonitored by detection of antibody titers in plasma or serum. StandardELISA, RIA or Western blots can be used with the Sir2 protein to assessthe levels of antibody.

In a preferred embodiment, the antibody, or antigen-binding fragment,selectively binds murine Sir2 proteins, such as mSir2α and mSir2β. An“antigen-binding fragment” of an antibody refers to an antibody whichbinds to any part of the Sir2 protein.

The antibodies of the invention can be human and nonhuman antibodies.The antibodies can be polyclonal, monoclonal, chimeric (e.g., humanchimeric antibodies), or fragments thereof. These antibodies can be usedto purify or identify the Sir2 protein contained in a mixture ofproteins, using techniques well known to those of skill in the art.These antibodies, or antigen-binding fragments thereof, can be used todetect the presence or absence of the Sir2 protein using standardimmunochemistry, Western blot methods, ELISA, or RIA. For example, andELISA and RIA assays can be used to quantitate the amount of Sir2 in acellular or nuclear extract.

As used herein, an “isolated” nucleic acid molecule (e.g., gene,nucleotide sequence, nucleic acid sequence), when referring to an Sir2protein encoded by a nucleic acid sequence, is a nucleic acid moleculewhich is not flanked by nucleic acid molecules which normally (e.g.,naturally occurring) flank the gene or nucleotide sequence (e.g., as ingenomic sequences) and/or has been completely or partially purified fromother transcribed sequences (e.g., as in a cDNA library). Thus, anisolated nucleic acid molecule, gene or nucleotide sequence can includea nucleic acid molecule, gene or nucleotide sequence which issynthesized chemically or by recombinant means. Nucleic acid moleculescontained in a vector are included in the definition of “isolated” asused herein. Also, isolated nucleic acid molecules include recombinantnucleic acid molecules and heterologous host cells, as well as partiallyor substantially or purified nucleic acid molecules in solution. In vivoand in vitro RNA transcripts of the invention are also encompassed by“isolated” nucleic acid molecules. Such isolated nucleic acid moleculesare useful for introduction into host cells, the manufacture of theencoded Sir2 protein, as probes for isolating homologues sequences(e.g., from other mammalian species or other organisms), for genemapping (e.g., by in situ hybridization), or for detecting the presence(e.g., by Southern blot analysis) or expression (e.g., by Northern blotanalysis, RNase protection assays) of related SIR2 genes in cells ortissue.

The present invention also encompasses isolated nucleic acid sequencesencoding the Sir2 proteins described herein, and fragments of nucleicacid sequences encoding biologically active Sir2 proteins. Fragments ofthe nucleic acid sequences, described herein, as useful as probes todetect the presence of SIR2. Specifically provided for in the presentinvention are DNA/RNA (also referred to herein as nucleic acid)sequences encoding mSir2α and mSir2β proteins, the fully complementarystrands of these sequences, and allelic variations thereof. Alsoencompassed by the present invention are nucleic acid sequences, genomicDNA, cDNA, RNA or a combination thereof, which are substantiallycomplementary to the nucleic acid sequences (e.g., DNA, RNA) encodingSir2 (e.g., mSir2α, mSir2β), and which specifically hybridize with theSIR2 nucleic acid sequences under conditions of stringency known tothose of skill in the art, those conditions being sufficient to identifynucleic acid sequences with substantial nucleic acid identity. Asdefined herein, substantially complementary means that the sequence neednot reflect the exact sequence of the SIR2 nucleic acid, but must besufficiently similar in identity of sequence to hybridize with SIR2nucleic acid under stringent conditions. Conditions of stringency aredescribed, for example, in Ausubel, et al., “Current Protocols inMolecular Biology”, John Wiley & Sons, Inc. (1999). Non-complementarybases can be interspersed in the sequence, or the sequences can belonger or shorter than SIR2 nucleic acid, provided that the sequence hasa sufficient number of bases complementary to SIR2 to hybridizetherewith.

The SIR2 nucleic sequence, or a fragment thereof, can be used as a probeto isolate additional SIR2 homologs. For example, a cDNA or genomic DNAlibrary from the appropriate organism can be screened with labeled SIR2nucleic acid probes (e.g., DNA or RNA) to identify homologous genes, asdescribed, for example, in Ausubel, F. M., et al., “Current Protocols inMolecular Biology”, John Wiley & Sons, (1999).

Typically the nucleic acid probe comprises a nucleic acid sequence ofsufficient length and complementary to specifically hybridize to nucleicacid sequences which encode mSir2α (SEQ ID NO: 25), mSir2β, mSir2γ,ySir2 or human Sir2. The requirements of sufficient length andcomplementarity can be easily determined by one of skill in the art.

Uses of nucleic acids encoding cloned Sir2 proteins or fragments includeone or more the following: (1) producing proteins which can be used, forexample, for structure determination, to further assess Sir2NAD-dependent deacetylation activity and/or mono-ADP-ribosyltransferaseactivity, or to obtain antibodies binding to the Sir2 protein; (2) todetermine the nucleotide sequence encoding a Sir2-like protein which canbe used, for example, as a basis for comparison with other proteins todetermine conserved regions, determine unique nucleotide sequences fornormal and altered proteins, and to determine nucleotide sequences to beused as target sites for antisense nucleic acids, ribozymes,hybridization detection probes, or PCR amplification primers; (3) ashybridization detection probes to detect the presence of a nativeprotein and/or a related protein in a sample; and (4) as PCR primers togenerate particular nucleic acid sequence regions, for example, togenerate regions to be probed by hybridization detection probes.

The Sir2 proteins and/or nucleic acid sequences include fragmentsthereof, (e.g., core domains of Sir2 proteins). Preferably, the nucleicacid contains at least 14, at least 20, at least 27, and at least 45,contiguous nucleic acids of a sequence provided in SEQ ID NO.: 25.Advantages of longer-length nucleic acid include producing longer-lengthprotein fragments having the sequence of a Sir2 protein which can beused, for example, to produce antibodies; increased nucleic acid probespecificity under high stringent hybridization assay conditions; andmore specificity for related the SIR2 nucleic acid under lowerstringency hybridization assay conditions.

Another aspect of the present invention features a purified nucleic acidencoding a Sir2 protein or fragment thereof. Specifically, mSIR2α (SEQID NO: 25), mSIR2γ and/or mSIR2β. Due to the degeneracy of the geneticcode, different combinations of nucleotides can code for the sameprotein. Thus, numerous protein fragments having the same amino acidsequences can be encoded for by difference nucleic acid sequences. Inpreferred embodiments, the nucleic acid encodes at least 12, at least18, or at least 54 contiguous amino acids of SEQ ID NO.: 25.

Another aspect of the present invention features a purified nucleic acidhaving a nucleic acid sequence region of at least 12 contiguousnucleotides substantially complementary to a sequence region in SEQ IDNO.: 25. By “substantially complementary” is meant that the purifiednucleic acid can hybridize to the complementary sequence region innucleic acid sequences of SEQ ID NO.: 25, under stringent hybridizingconditions. Such nucleic acid sequences are particularly useful ashybridization conditions, only highly complementary nucleic acidsequences hybridize. Preferably, such conditions prevent hybridizationof nucleic acids having 4 mismatches out of 20 contiguous nucleotides,more preferably 2 mismatches out of 20 contiguous nucleotides, mostpreferably one mismatch out of 20 contiguous nucleotides. In preferredembodiments, the nucleic acid is substantially complementary to at least20, at least 27, or at least 45 contiguous nucleotides provided in SEQID NO.: 25.

Another aspect of the present invention features a purified proteinhaving at least about 5-20 contiguous amino acids of an amino acidsequence provided in SEQ ID NOS.: 1, 2, 3, 4, 5, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27 or 28. By “purified” inreference to a protein is meant that the protein is in a form (e.g., itsassociation with other molecules) distinct from naturally occurringprotein. Preferably, the protein is provided as substantially purifiedpreparation representing at least 75%, more preferably 85%, mostpreferably 95% or the total protein in the preparation. In preferredembodiments, the purified protein has at least 5 contiguous amino acidsof SEQ ID NO.: 27 or 28.

Preferred protein fragments include those having functional Sir2activity (e.g., alter the NAD-dependent acetylation status of histoneproteins such as H3 or H4; mono-ADP-ribosylate nuclear proteins such asH2B, H3 or H4), or epitope for antibody recognition. Such proteinfragments have various uses such as being used to obtain antibodies to aparticular region and being used to form chimeric proteins withfragments of other proteins create a new protein having uniqueproperties, such as a longer half-life.

Nucleic acid molecules can be inserted into a construct which can,optionally, replicate and/or integrate into a recombinant host cell, byknown methods. The host cell can be a eukaryote or prokaryote andincludes, for example, yeast (such as Pichia pastorius or Saccharomycescerevisiae) bacteria (such as Escherichia coli or Bacillus subtilis),animal cells or tissue, insect Sf9 cells (such as baculoviruses infectedSF9 cells) or a mammalian cells (somatic or embryonic cells, Chinesehamster ovary cells, HeLa cells, human 293 cells, monkey COS-7 cells andmouse NIH 3T3 cells).

The invention also provides vectors or plasmids containing one or moreof each of the SIR2 genes. Suitable vectors for use in eukaryotic andprokaryotic cells are known in the art and are commercially available orreadily prepared by a skilled artisan. Additional vectors can also befound, for example, in Ausubel, F. M., et al., “Current Protocols inMolecular Biology”, John Wiley & Sons, Inc. (1999) and Sambrook et al.,“Molecular Cloning: A Laboratory Manual,” 2nd Ed. (1989), the teachingsof which are incorporated herein by reference in their entirety.

Another aspect of the present invention features a recombinant host cellcomprising an isolated nucleic acid molecule of a SIR2 gene operablylinked to a regulatory sequence. “Operably linked” is intended to meanthat the nucleotide sequence(s) is linked to a regulatory sequence in amanner which allows expression of the nucleic acid sequence(s). Therecombinant host cell is made up of an isolated nucleic acid sequenceencoding at least about 5-20 contiguous amino acids provided in SEQ IDNO.: 1, 2, 3, 4, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 26, 27 or 28 and a cell able to express the nucleic acid.Cells containing a functioning Sir2 protein can be used, for example, toscreen to agonists or antagonists of Sir2 activity.

The nucleic acid molecule can be incorporated or inserted into the hostcell by known methods. Examples of suitable methods of transfecting ortransforming cells include calcium phosphate precipitation,electroporation, microinjection, infection, lipofection and directuptake. “Transformation” or “transfection” as used herein refers to theacquisition of new or altered genetic features by incorporation ofadditional nucleic acids, e.g., DNA. “Expression” of the geneticinformation of a host cell is a term of art which refers to the directedtranscription of DNA to generate RNA which is translated into a protein.Methods for preparing such recombinant host cells and incorporatingnucleic acids are described in more detail in Sambrook et al.,“Molecular Cloning: A Laboratory Manual,” 2nd Ed. (1989) and Ausubel, etal. “Current Protocols in Molecular Biology,” (1999), for example.

The host cell is then maintained under suitable conditions forexpression of Sir2. Generally, the cells are maintained in a suitablebuffer and/or growth medium or nutrient source for growth of the cellsand expression of the gene product(s). The growth media are not criticalto the invention, are generally known in the art and include sources ofcarbon, nitrogen and sulfur. Examples include Luria broth, Superbroth,Dulbecco's Modified Eagles Media (DMEM), RPMI-1640, M199 and Grace'sinsect media. The growth Media may contain a buffer, the selection ofwhich is not critical to the invention. The pH of the buffered Media canbe selected and is generally one tolerated by or optimal for growth forthe host cell.

The mSIR2α gene can be expressed in mammalian or insect cells, anymammalian or insect expression plasmid or viral vector driven by nativemSIR2α promoter can be used, any of other mammalian or insect promotersand enhances, any of viral promoters, or any of artificial induciblepromoters can be used. The host cells can be cells of murine, human, orany of other organisms including insects cultured in conventional orspecialized in vitro culture conditions. The cells transfected with amSIR2 expression plasmid or infected by the virus containing a mSIR2αexpression vector.

The host cell is maintained under a suitable temperature and atmosphere.Alternatively, the host cell is aerobic and the host cell is maintainedunder atmospheric conditions or other suitable conditions for growth.The temperature should also be selected so that the host cell toleratesthe process and can be, for example, between about 13°-40° C.

The invention also relates to a method of preparing a Sir2 protein usingthe recombinant host cells described above. The recombinant host cellsare biological factories to produce proteins encoded for by the isolatednucleic acid molecule of SIR2.

The chromosomes of higher eukaryotes are compartmentalized into twocytogenetically distinct regions, euchromatin and heterochromatin.Heterochromatin was initially described as chromosomal regions thatfailed to decondense after mitosis (Heitz, E. Jb. Wiss. Bot. 69:728(1928)) Subsequent studies have revealed that heterochromatin is formedon highly repetitive DNA, such as centromeres and telomeres, replicateslate in the cell cycle and is transcriptionally inactive (Hennig, W.Heterochromatin. Chromosoma 108:1-9 (1999)). Although many questionsabout the functional and structural properties of heterochromatin areunresolved, progress in understanding its molecular nature has been madein studies of yeast (Karpen, G., et al., Trends Genet. 13: 489-496(1997)); (Grunstein, M. Cell 93, 325-328 (1998)) and Drosophila(Wakimoto, B. T., Cell 93: 321-324 (1998)). In Drosophila, genesjuxtaposed to centromeric heterochromatin through chromosomalrearrangement or transposition become inactivated. The phenomenon, knownas position effect variegation (PEV) (Muller, H. J.; J. Genet. 22:299-335 (1930)), results from the spreading of heterochromatin acrossthe rearrangement breakpoint. Genetic screens have identified largenumbers of PEV modifiers some of which have been cloned andcharacterized (Reuter, G., et al., BioEssays 14: 605-612 (1992);(Weiler, K. S., et al., Annu. Rev. Genet. 29: 577-605 (1995); (Wallrath,L. L., Curr. Opin. Genet. Dev. 8: 147-153 (1998)). Heterochromatinprotein 1 (HP1) are one of the best-characterized heterochromatincomponents and known to localize at centromeres and telomeres (James, T.C., et al., Mol. Cell. Biol. 6: 3862-3872 (1986); (James, T. C., et al.,Eur. J. Cell Biol. 50: 170-180 (1989); (Fanti, L., et al., Mol. Cell. 2:527-538 (1998)). HP1-related proteins have been identified from a widevariety of organisms including mammals, and a region termed thechromodomain is highly conserved in HP1 family members (Singh, P. B., etal., Nucleic Acids Res. 19: 789-794 (1991)). The chromodomain is alsoshared by a related set of chromatin factors, the Polycomb group (Pc-G)(Pirrotta, V., Cell 93: 333-336 (1998)). Pc-G is required to maintainthe repressed state of homeotic genes through many cell divisions in thedevelopmental stages of Drosophila (Orlando, V., et al., Cell 75: 1187(1993)).

Transcriptional silencing in budding yeast is associated with genomicdomains of structurally altered chromatin akin to heterochromatin inhigher cells (Sherman, J. M., et al., Trends Genet. 13: 308-313 (1997)).

The principle genes required for silencing SIR2, SIR3, and SIR4 encodemembers of a complex that is targeted to the specific chromosomaldomains that are silenced (Ivy, J. M., et al., Mol. Cell. Biol. 6:688-702 (1986); (Gotta, M., et al., J. Cell Biol. 134: 1349-1363 (1996);(Rine, J., et al., Genetics 116: 9-22 (1987); (Aparicio, O. M., et al.,Cell 66: 1279-1287 (1991)). For example, silencing at the extra copiesof a and a mating type genes at HMLα and HMRa is mediated by the bindingof these three Sir proteins, plus a fourth, Sir1p, to a set of DNAbinding factors including Rap1p, ORC, and Abf1p (Triolo, T., et al.,Nature 381: 251-253 (1996)).

In contrast, at telomeres Sirs 2p, 3p, and 4p bind to tandem Rap1pmolecules arrayed at the telomeric repeat tract (Hardy, C. F. J., etal., Genes Dev. 6: 801 (1992); (Moretti, P., et al., Genes Dev. 8: 2257(1994)). Finally, at the rDNA, Sir2p, but not the other Sir proteins, isbound to a specific region in the rDNA spacer in a complex termed RENTwhich includes another protein, Net1p (Shou, W., et al., Cell 97:233-244 (1999)).

The function of SIR-mediated silencing at HM loci is to prevent theexpression of both a and α mating types, which would lead to non-mating,pseudo-diploid strains. The role of the Sir complex at telomeres is lessapparent. Interestingly, the telomeric Sir complex, along with the Kuprotein, moves to sites of double strand DNA breaks to aid their repairby the non-homologous end joining pathway, suggesting that telomeric Sircomplex exists as a poised DNA repairosome (Mills, K. D., et al., Cell97: 609-620 (1999); (Martin, S. G., et al., Cell 97: 621-633 (1999)).

Sir2p in the rDNA silences marker genes inserted there (Bryk, M., etal., Genes Dev. 11: 255-269 (1997); (Smith, J. S., et al., Genes Dev.11: 241-254 (1997)) and, plays an important role in repressingrecombination within the 100-200 tandemly repeated copies of the rDNAsequences in chromosome XII (Gottlieb, S., et al., Cell 56: 771-776(1989)).

Replicative life span in yeast mother cells (the number of times amother cell divides to give daughters) is limited, at least in part, bythe accumulation of extrachromosomal rDNA circles (ERCs) in nuclei ofaging mothers (Sinclair, D. A., et al., Science 277: 1313-1316 (1997)).These accumulated ERCs result from the excision of circles from thechromosomal rDNA array by homologous recombination (Park, P. U., et al.,Mol. Cell. Biol. 19: 3848-3856 (1999)), the subsequent replication ofERCs in future cell divisions, and the preferential segregation of ERCsto mother cells.

SIR2, SIR3, and SIR4 play a key role in determining the life span ofyeast mother cells (Kennedy, B. K., et al., Cell 89: 381-391 (1997);(Kaeberlein, M., et al., Genes Dev. 13: 2570-2580 (1999)). Deleting SIR3or SIR4 shortens the life span by about 25%, and deleting SIR2 shortensthe life span by about 50%. The shortening in the sir3 and sir4 mutantsis due to the simultaneous expression of a and α mating typeinformation, which somehow causes an increase in recombination in therDNA leading most likely to an earlier accumulation of a lethal numberof ERCs (Kaeberlein, M., et al., Genes Dev. 13: 2570-2580 (1999)).

The shortening in sir2 mutants is due to the absence of Sir2p in therDNA leading to the elevated rate of recombination at that locus and therapid generation of ERCs. The short life span of sir2 mutants issuppressed by deletions of FOB1 (Kaeberlein, M., et al., Genes Dev. 13:2570-2580 (1999)), which encodes a protein that creates a unidirectionalbarrier to DNA replication in the rDNA (Kobayashi, T., et al., GenesCells 1: 465-474 (1996)). Evidently this barrier is a signal thatprovokes recombination in the rDNA and the generation of ERCs (Defossez,P. A., et al., Mol. Cell. 3: 447-455 (1999)), and the role of Sir2p isto counteract this hyperrecombination. In the absence of Fob1p (and thereplication barrier) Sir2p is no longer needed to prevent the formationof ERCs. The addition of a second copy of SIR2 to cells extends thereplicative life span of mothers well beyond that of the wild type,indicating that Sir2p is the limiting factor for life span (Kaeberlein,M., et al., Genes Dev. 13: 2570-2580 (1999)).

Since chromatin at HML and HMR is hypoacetylated (Braunstein, M., etal., Mol. Cell. Biol. 16: 4349-4356 (1996); (Braunstein, M., et al.,Genes Dev 7: 592-604 (1993)), Sir2p was hypothesized to be a histonedeacetylase. However, it is also possible that hypoacetylation ofchromatin results from some other effect, such as the exclusion ofacetylases by the inaccessible structure of these chromosomal regions orthe recruitment of other deacetylases.

More recent studies identified SIR2 orthologs in yeast and homologs inmany species, including bacteria, which all share a core homology ofabout 270 amino acids. The four S. cerevisiae orthologs HST1-4 do notplay any role in yeast silencing, with the exception that high levelexpression of HST1 can partially compensate for the absence of SIR2(Brachmann, C. B., et al., Genes Dev. 9: 2888-2902 (1995)). Thebacterial homolog, cobB, can apparently function in cells in abiosynthetic step that transfers phosphoribose from nicotinatemononucleotide to dimethyl benzimidazole (Tsang, A. W., et al., J. Biol.Chem. 273: 31788-31794 (1998)). The present invention documents thatyeast Sir2p and its mouse homologue mSir2α aremono-ADP-ribosyltransferases and NAD hydrolases with specificallyacetylated histone substrates. Amino acid changes in mSir2α that disruptthis enzymatic activity also reduce function in vivo. The modificationof the acetylation status of N-terminal tails of histones by Sir2 cantrigger genomic silencing in yeast and heterochromatin formation moregenerally in eukaryotic organisms.

As described herein, yeast Sir2p and its mouse Sir2α homologue possessADP-ribosyltransferase and NAD hydrolase. These activities arestimulated by N-terminal peptides of histones H3 and H4. In particular,acetylated lysines of histone proteins Lys9Ac and Lys14Ac of H3 andLys16 of the H4. Transfer to histone H2B is also observed. Studies usingenzyme inhibitors and snake venom phosphodiesterase show that Sir2p andmSir2α are mono-ADP ribosyltransferases.

Sir2 Proteins are Novel Nuclear Mono-ADP-Ribosyltransferases withHistone Substrates

Yeast and mouse Sir2 proteins are shown herein to bemono-ADP-ribosyltransferases by several criteria. First, the proteinstransfer ³²P-labeled ADP-ribose from NAD to histones H2B and H3. Theendogenous mSir2α protein immunoprecipitated from NIH3T3 cell extractsshows the same activity. Second, this modification can be removed bysnake venom phosphodiesterase, which digests the phosphodiester bond inthe ADP-ribose moiety. Third, the reactions are sensitive tomono-ADP-ribosylation inhibitors, but not to poly(ADP-ribose)polymerase(PARP) inhibitors. ySir2p and mSir2α provide the first examples ofnuclear mono-ADP-ribosyltransferases in yeast and mammals.

In the mammalian nucleus, PARPs are likely involved in DNA repair(Menissier-de Murcia, J., et al., Proc. Natl. Acad. Sci. USA 94:7303-7307 (1997); (Wang, Z. Q., et al., Genes Dev. 11: 2347-2358(1997)), replication (Yoshida, S., et al., Mol. Cell. Biochem. 138:39-44 (1994)), transcription (Oliver, F. J., et al., Embo J. 18:4446-4454 (1999)), apoptosis (Germain, M., et al., J. Biol. Chem. 274:28379-28384 (1999)) and telomere maintenance (Di Fagagna, F. D. A., etal., Nat. Genet. 23: 76-80 (1999)). On the other hand, it has beenreported that alkylating agents induce mono-ADP-ribosylation of histonesH1, H2B, and H3 (Kreimeyer, A., et al., J. Biol. Chem. 259: 890-896(1984); (Adamietz, P., et al., J. Biol. Chem. 259:6841-6846 (1984)).Interestingly, many viral, bacterial, and eukaryotic enzymes such asdiphtheria, pertusis, and cholera toxins (DT, PT, and CT, respectively)and vertebrate arginine-specific mono-ADP-ribosyltransferases catalyzesimilar reactions (Domenighini, M., et al., Mol. Microbiol. 21: 667-674(1996)).

These ADP-ribosyltransferases share structural motifs in their catalyticdomains and can be classified into two groups: the DT group and the CTgroup (Domenighini, M., et al., Mol. Microbiol. 21: 667-674 (1996);(Moss, J., et al., Mol. Cell. Biochem. 193: 109-113 (1999)). The DTgroup includes diphtheria toxin and the family of PARPs, while the CTgroup includes CT, PT, and other eukaryoticmono-ADP-ribosyltransferases. ySir2p and mSir2α share at least one motifof the CT group (see FIG. 2C), hydrophobic-Ser-Thr-Ser-hydrophobic(VSTSL, SEQ ID NO: 30, in ySir2p and VSVSC, SEQ ID NO: 31, in mSir2α),which forms the NAD-binding cleft. The mutation of the first serine,which is known to be important for positioning NAD, abolishes themono-ADP-ribosylation activity of the mSir2α (see FIG. 6C).

The mutagenesis of highly conserved residues in the mSir2α core domainrevealed that mutations in 10 of 10 residues affected themono-ADP-ribosylation activity. In mouse cells, 9 of these 10 mutantswere inactive in a transient assay for repression of a reporter,suggesting that the enzymatic activity is critical in vivo. The mutant,P285A, could misfold in E. coli and thus, be inactive. Genetic analysisof the core of yeast Sir2p indicates that the four conserved cysteinesat positions 372, 374, 396, and 398 are crucial for silencing in vivo.Further, chimeras in which a human Sir2 core domain is substituted forthe yeast domain can function in silencing at HM loci, but not attelomeres or rDNA.

Specific Acetylation of H3 and H4 Tails Stimulates Sir2 Activities

A striking feature of the specificity of ADP-ribosylation of H3 wasrevealed by the use of N-terminal peptides (residues 1-20). Sir2modified the H3 N-terminal tail peptide when Lys9 and Lys 14 wereacetylated but not when the peptide was unacetylated. These lysines areacetylated and important for silencing in vivo (Thompson, J. S., et al.,Nature 369: 245-247 (1994)). In addition, a large fraction of the NADwas hydrolyzed H specifically in the presence of the diacetylatedpeptide and either yeast or mouse Sir2. This latter activity can reflectinefficient transfer of ADP-ribose to the peptide substrate or canpresent another yet unknown activity of Sir2 proteins in themodification of histones. These data show that the N-terminal tail of H3is a biologically relevant target of Sir2 proteins in vivo.

An H4 peptide (residues 1-20) acetylated on Lys16 stimulated bothADP-ribosyltransferase and NAD hydrolase activities of Sir2, but H4peptides acetylated on Lys5, Lys8, or Lys12 did, at best, very weakly. Atetra-acetylated H4 peptide (acylated Lys5, 8, 12 and 16) was also apoor substrate. Lys16 is a highly acetylated residue in mammalianchromatin. In constitutive heterochromatin, H4 is hypoacetylated at allfour lysines, but in facultative heterochromatin Lys5, 8, and 12 arehypoacetylated, while Lys16 is not. Thus, these data illustrate the roleof Sir2 in regulating the state of facultative heterochromatin,consistent with the observed intracellular localization of the protein.In yeast mutations of Lys16 to an uncharged residue, glutamine, reducesilencing, and mutation to arginine, even though positive charge isretained, also reduces silencing. Mutations in Lys5, 8, or 12 toarginine, in contrast, exert smaller effects on silencing. Thesefindings are consistent with the idea that the acetylated Lys 16 residueof H4 is critical for recognition by Sir2.

ADP-ribosylation of histone N-terminal tails can lead to heterochromatinand gene silencing by interacting with histone residues that areconstitutively acetylated, like Lys16 of mammalian H4, thereby inducingADP-ribosylation by Sir2 proteins in one or more adjacent residues inthe N-terminal tail (FIG. 7). In active chromatin, the lysines in theN-terminal tails of H3 and H4 are acetylated (depicted by stars at top).Sir2 recognizes H3 and H4 tails containing specifically acetylatedlysines (indicated by a large star). Sir2 ADP-ribosylates theseN-terminal tails, causing the formation of heterochromatin, perhaps bythe recruitment of histone deacetylases. The ADP-ribosylation in turnfacilitates silencing, for example, by restricting access of histoneacetylases or by recruiting deacetylases, leading to the hypoacetylatedstate of other lysine residues in the tail and thus a tighter chromatinstructure. ADP-ribosylation and deacetylation are targeted to specificregions of the genome by the selective recruitment of Sir2. In yeast,Sir3p and Sir4p facilitating targeting of Sir2p to telomeres and HMloci, and Net1p and perhaps other components of the RENT complex recruitSir2p to the rDNA. Consistent with this model, overexpression of Sir2induces a more global hypoacetylation of histones H2B, H3 and H4 inyeast.

The model depicted in FIG. 7 is consistent with the silencing phenotypesof several yeast mutants. Rpd3p is a histone deacetylase. Rpd3 mutantsshow hyperacetylation of histones and generally an increase intranscription of genes assayed. An exception to this rule, however,occurs in the case of silenced chromatin at yeast telomeres and rDNA,where silencing actually increases in the mutant. The increase insilencing can occur as a result of hyperacetylation of the lysines inthe N-terminal tail that are recognized by Sir2. NAT1/ARD1 encodesubunits of an N-terminal acetyltransferase. Mutations in either geneabolish silencing in yeast. It is possible that the nat1 or ard1 mutantshave underacetylation of histone residues that potentiate Sir2 activity.Additionally, or alternatively, the effects of these mutants can beindirect.

Mammalian Sir2 Proteins

SIR2 homologs have been found in a variety of organisms. Human SIR2homologs that all correspond to the same gene, mSIR2β, which is mostrelated to yeast HST2 have been reported (Afshar, et al., Gene234:161-168 (1999); Frye, Biochem. Biophys. Res. Commun. 260:273-279(1999); Sherman, et al., Mol. Biol. Cell 10:3045-3059 (1999)). Incontrast, the human SIR2 gene appears to correspond to a fragment of ahomolog of mSIR2α. Of all mammalian SIR2 homologs analyzed to date,mSIR2α shows the greatest homology to ySir2p in the evolutionarilyconserved core domain. Nonetheless, there are some significantdifferences between ySir2p and mSir2α. mSir2α has a diverged N-terminalportion and a longer C-terminal tail and is almost twice the size ofySir2p. It is possible that these mSir2α-specific regions may beinvolved in species-specific or cell type-specific protein-proteininteractions.

In yeast, the Sir complex moves to sites of DNA double strand breaks.Since ADP-ribosylation has been associated with sites of DNA repair inmammalian cells, it is likely that mammalian Sir2 proteins are recruitedto DNA damage. Mono-ADP-ribosylation of histones near sites of DNAdamage in yeast and mammals likely have an important biological role.

Link Between Energy Metabolism, Heterochromatin and Aging

The substrate for Sir2 modification of chromatin, NAD, may be anindicator of the energy status of cells. Cells grown under energylimitation may have a high NAD/NADH ratio, while cells growing underenergy surplus would have a low ratio. Since NAD but not NADH isexpected to function as a substrate in the mono-ADP-ribosylationreaction, Sir2 activity can be highest under conditions of energylimitation. Thus, the rate of energy metabolism may control geneexpression of certain chromosomal domains by regulating the activity ofSir2.

Recent studies indicate that Sir2p is a limiting component in thedetermination of the life span of yeast mother cells for example,overexpression of Sir2p extends the life span beyond wild type. Caloricor dietary restriction is associated with an extension of life span inrodents, C. elegans, and even yeast. Caloric restriction may exert ofits effect, in part, by creating a condition of energy limitation, whichwould activate Sir2 and promote ADP-ribosylation of chromatin and genesilencing. ADP-ribosylation is a biochemical event which has beensuggested to play a role in aging. Alterations in the maintenance ofSir2-silenced chromatin could result in normal mammalian aging and byslowing caloric restriction. The fact that Sir2 proteins are simpleenzymes suggests a strategy for intervening in non genetic ways toinfluence life span. Small molecules that are agonists for this enzymecan extend life span in yeast cells and in mammals.

The present invention shows that yeast Sir2p and its mouse homologmSir2α are ADP-ribosyltransferases and NAD hydrolases with specifichistone substrates, the N-terminal tails of H3 and H4. Both activitiesare stimulated by acetylation in the histone tails; in particular Lys9Acand Lys14Ac for H3, and Lys16Ac for H4. Acetylation of these lysineresidues is important for silencing of chromatin in vivo. Mutations inthe conserved sequences of mSir2α reduce ADP-ribosyltransferase activityin vitro and chromatin of the silencing activity in mouse NIH3T3 cells.The mSir2α protein is localized in the nuclei of mouse cells shown thatacetylation-terminal tails of H3 and H4 trigger ADP-ribosyltransferaseand NAD hydrolase activities of Sir2 proteins. The ADP-ribosylation andacetylation of histones proteins can be important in the formation ofheterochromatin in eukaryotic cells.

The present invention shows that Sir2 proteins are novel NAD-dependentdeacetylases specific for the N-terminal tail of histone H3. AlthoughNAD and NADH are frequent enzyme cofactors in oxidation/reductionreactions, this is the first time NAD has been shown to drive a distinctenzymatic reaction in a substrate, such as a peptide of theamino-terminal tail of histone H3 di-acetylated at lysines 9 and 14.

Since histones in silenced chromatin are hypoacetylated, and becauseoverexpressed Sir2p promotes global deacetylation of histones in yeast(Braunstein, M., et al., Genes Dev 7: 592-604 (1993); (Braunstein, M.,et al., Mol. Cell. Biol. 16: 4349-4356 (1996)), it was originallyproposed that Sir2p might be a histone deacetylase. However, such anactivity could not be demonstrated until now, presumably because of itsabsolute requirement for NAO. The present invention is consistent withstudies that show mutating lysines in the tails of histones H3 and H4can reduce silencing in vivo (Thompson, J. S., et al., Nature 369:245-247 (1994); (Braunstein, M., et al., Mol. Cell. Biol. 16: 4349-4356(1996)) and suggest that this NAD-dependent deacetylation of histonesper se is sufficient to establish silencing in vivo. These data showthat SIR2 mutations reduce or eliminate deacetylase activity showcorresponding defects in silencing, suppression of recombination, andlife span in vivo. The nature of the requirement of NAD fordeacetylation is not yet clear, but at the concentrations of about onehundred μM, NAD may serve as an allosteric effector to activate thehistone deacetylase activity of Sir2. The failure of NADH, NADP, orNADPH to function as such reveals a remarkable specificity of Sir2p forthe oxidized form of NAD. Sir2 proteins catalyze the putative transferof ADP-ribose from NAD to the proteins such as intact histone H3 or theamino-terminal peptide and labeled NAD, to allow the detection of lowlevels of ³²P transfer. However, in reactions allowing a directcomparison between ADP-ribosylation and deacetylation (using the H3peptide at 1 mM NAD), the reaction products displayed at least 27%conversion of the peptide to deacetylated species, and no detectableconversion to any ADP-ribosylated species. Thus, ADP ribosyltransferasereaction proceeds to a much lesser degree than deacetylation, at leastwith this H3 peptide substrate.

Coumermycin A1 is an inhibitor of mono-ADP-ribosyltransferases (Banasik,M., et al., Mol. Cell. Biochem. 138: 185-187 (1994)) and was shown toinhibit the putative ADP-ribose transferase activity as well as theaccompanying NAD hydrolysis. However, coumermycin A1 did not inhibit theSir2p deacetylase activity at all. These findings indicate that theputative ADP-ribosyltransferase activity is separable and may play adistinct role in vivo. The ADP-ribosylation of histones is known tooccur when cells are treated with DNA damaging agents (Adamietz, P., etal., J. Biol. Chem. 259:6841-6846 (1984); (Kreimeyer, A., et al., J.Biol. Chem. 259: 890-896 (1984); (Pero, R. W., et al., Mutat. Res. 142:69-73 (1985)). Moreover, antibodies against mono-ADP-ribose react withmammalian nuclei only if when cells are treated with DNA-damagingagents. Thus ADP-ribosylation of histones and NAD hydrolysis may play arole in opening up chromatin to allow DNA repair. In this regard, theSir proteins are known to move to sites of DNA breaks to aid theirrepair by non-homologous end joining (Mills, K. D., et al., Cell 97:609-620 (1999); (Martin, S. G., et al., Cell 97: 621-633 (1999)).

There are four SIR2-related genes in yeast, HST1-4 (Brachmann, C. B., etal., Genes Dev. 9: 2888-2902 (1995)), but no single hst mutant affectssilencing, although effects are observed in the hst3/hst4 double mutant.It is expected that the these gene products catalyze histonedeacetylation. In mammals there are at least five homologs (Frye, R. A.,Biochem. Biophys. Res. Commun. 260: 273-279 (1999); (Brachmann, C. B.,et al., Genes Dev. 9: 2888-2902 (1995)), of which mSir2α is the mostclosely related to Sir2p. mSir2α is identical to yeast Sir2p in thedeacetylation reaction, indicating that this activity is highlyconserved in nature.

mSir2α is not at the rDNA, centromeres, or telomeres, but broadlydistributed in nuclei (not shown) suggesting that Sir2 proteins mayregulate silencing in widely distributed blocks of the mammalian genome.A distinction between Sir2-silenced heterochromatin and constitutiveheterochromatin is illustrated by the disruption of centromericheterochromatin in S. pombe by the general deacetylase inhibitor,trichostatin A (TSA) (Ekwall, K., et al., Cell 91: 1021-1032 (1997)),which does not affect the deacetylase activity of Sir2p. The involvementof a distantly related SIR2 family member, hst4+, in this centromericsilencing (Freeman-Cook, L. L., et al., Molecular Biology of the Cell10: 3171-3186 (1999)) provides a hint that the more distant SIR2 familymembers may target constitutive heterochromatin, while closely relatedSIR2 genes target regulated blocks of silencing.

In addition to a role in silencing, Sir2p also extends life span ofmother cells by repressing recombination in the rDNA (Gottlieb, S., etal., Cell 56: 771-776 (1989)), and reducing the production of toxic rDNAcircles (Sinclair, D. A., et al., Science 277: 1313-1316 (1997);(Sinclair, D. A., et al., Cell 91: 1-20 (1997)). The fact that NAD butnot NADH, NADP, or NADPH can activate deacetylation of histones by Sir2pmay link chromatin silencing and ageing to the metabolic state of cells,i.e., Sir2 proteins can sense the energy or oxidation state of cells.Most of the NAD in eukaryotic cells is present in the nucleus(Rechsteiner, M., et al., Nature 259: 695-696 (1976)), where it turnsover rapidly (Dai, Y., et al., Mut. Res. 191: 29-35 (1987)). Thus, apool of NAD can regulate chromatin structure by controlling the histonedeacetylase activity of Sir2 proteins.

It is interesting to note that caloric restriction slows ageing in avariety of organisms, including yeast (Muller, I., et al., Mech. AgeingDev. 12: 47-52 (1980)), C. elegans (Lakowski, B., et al., Proc. Natl.Acad. Sci. USA 95: 13091-13096 (1998)), rodents (Weindruch, R. H., etal., J. Nutrit. 116: 641-654 (1986)), and probably primates (Roth, G.S., J. Am. Geriatr. Soc. 47: 896-903 (1999)). It is possible that thealtered metabolic rate in calorically restricted cells exert at leastpart of its effect by increasing the availability of NAD in the nucleus,which, in turn, up-regulates Sir2 proteins and chromatin silencing. Thehunkering down of the genome in the face of carbon limitation issensible because it conserves energy and could explain the extended lifespan under this regimen. In normal ageing, NAD levels decline (Chapman,M. L., et al., Mech. Of Aging and Dev. 21: 157-167 (1983)), perhapscausing a decrease in Sir2 activity and a deleterious loss of genomicsilencing.

It is clear that nuclei obtained from adult animals may be reprogrammedin oocytes to undergo embryonic development and produce viable progeny(Wilmut, I., et al., nature 385: 810-813 (1997); (Wakayama, T., et al.,Nature 394: 369-374 (1998)). This cloning implies that there can be noirreversible changes in nuclei of ageing animals, such as DNA deletionsor DNA circles. Where changes in chromatin structure the basis ofageing, then such a reversibility in oocytes would be theoreticallypossible. It is possible that reprogramming of adult nuclei, a slow andinefficient process, can be facilitated by the addition of chromatinmodification factors, such as Sir2 to oocytes. Moreover, it possiblethat increasing the activity of Sir2 in metazoans extends their lifespan, as it does in yeast (Kaeberlein, M., et al., Genes Dev. 13:2570-2580 (1999)). The most ready way to achieve this goal is to provideadditional copies of SIR2 genes in transgenic animals. A more long termstrategy with possible implications to humans is to intervene with smallmolecules that increase the deacetylation activity of Sir2. The latterapproach offers the advantage of late intervention to avoid any unwantedside effects on development or fertility and could, in principle, affordthe benefits of caloric restriction without the practical difficulties.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. The teachings of all referencescited herein are hereby incorporated by reference in their entirety.

EXEMPLIFICATION Materials and Methods

Data Base and Sequence Analysis

TBLASTN searches were performed on the NCBI mouse EST sequencedatabases, using the amino acid sequence of ySir2p. All mouse ESTsequences homologous to ySir2p were classified into three groups termedα, β, and γ, based on the homology results from the searches. Threerepresentative EST cDNA clones were purchased for three mouse homologgroups from GENOME SYSTEMS, INC. (St. Louis, Mo.): AA199012 for α,AA105536 for β, and AA260334 for γ. The cDNA clones were partially orcompletely sequenced. All deduced amino acid sequences were aligned withthe CLUSTAL X program. To cover each core domain completely, amino acidsequences of AA137380 and AA212772 for β and γ respectively, were alsoused. A phylogenetic tree of the core domains of the yeast and mouseSir2 families was generated with the CLUSTAL X and NJPLOT program byusing the following amino acid sequences: position 228-499 for ySir2p,174-440 for yHst1p, 1-251 for yHst2p, 26-315 for yHst3p, 65-343 foryHst4, 215-460 for mSir2α, TLGL to LINKEK (SEQ ID NO: 32) for mSir2β,and FGGG to LINRDL (SEQ ID NO: 33) for mSIR2γ.

Northern Blotting

The multiple tissue Northern blot (heart, brain, spleen, lung, liver,skeletal, muscle, kidney, testis) was purchased from CLONTECH™ (PaloAlto, Calif.). Prehybridization and hybridization were performed at 65°C. in 5×SSPE, 5×Denhardt's solution, 1% SDS and 0.1 mg/ml poly(A). ThecDNA fragments of AA199012, AA105536, and AA260334 were used as probesfor mSIR2α, β, and γ, respectively. The human β actin fragment from themanufacturer was used as a control probe. Between each blotting, theprobe was stripped off by boiling the membrane in 0.5% SDS for 1 min.

Molecular Cloning of mSIR2α

The mouse 15-day embryo 5′ STRETCH PLUS™ cDNA library (CLONTECH™) wasscreened with the cDNA fragment of AA199012 as a probe. Five positiveclones were obtained from approximately one million independent plaques.One of the five clones contained a 3.9 kb cDNA fragment. The nucleotidesequence of this fragment was determined with an Applied Biosystems 374automated sequencer. Although more 5′ sequences of mSIR2α cDNA wereobtained by 5′ RACE with the mouse liver Marathon-Ready cDNA(CLONTECH™), no stop codon could be found in the upstream of the firststart codon (data not shown). This finding was confirmed on the genomicsequence of the mSIR2α gene (data not shown). Thus, the cDNA cloneencodes the full-length mSir2α protein, as shown in FIG. 2B. The deducedamino acid sequence of mSir2α was aligned in the CLUSTAL X and SEQVU 1.1programs with other Sir2 family members.

Antibody Production to mSIR2α

The 5′ SalI-PvuI fragment of the mSIR2α cDNA was engineered to be clonedinto BamHI site of pET16b vector (NOVAGEN®, Madison, Wis.). The BL21(DE3) pLysS bacterial strain that also has an extra copy of argininetRNA gene was transformed with the resultant plasmid. A transformedbacterial clone was induced in 1 mM IPTG at 37° C. for 7 hrs to produce10×His-tagged N-terminal fragment of the mSir2α protein. The mSir2αN-terminal protein was purified with Ni-NTA agarose (QIAGEN®, Valencia,Calif.) under denaturing condition. Rabbit polyclonal antisera againstthis purified protein were produced at COVANCE RESEARCH PRODUCTS INC.(Denver, Pa.). Affinity purification of the antibody was performed withHiTrap NHS-activated column (AMERSHAM PHARMACIA BIOTECH INC.,Piscataway, N.J.) conjugated with the dialyzed mSir2α N-terminalprotein.

Western Blotting and Immunoprecipitation

Mouse NIH3T3 cells were lysed directly in Laemmli's sample buffer forWestern blotting or in extraction buffer (20 mM Tris-HCl [pH7.6], 150 mMNaCl, 0.5% IGEPAL CA-630 (SIGMA®, St. Louis, Mo.), 1 mM EDTA, 0.5 mMPMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin) forimmunoprecipitation. The in vitro translated mSir2α protein was producedwith the XbaI-linearized PBLUESCRIPT® containing the mSIR2α cDNA at SalIsite and the STP3 T7 in vitro transcription/translation kit (NOVAGEN®).For Western blotting, 17 μg of the extract or 2 μl of the in vitrotranslation mixture was run on a 4-15% gradient SDS-PAGE gel andtransferred onto an IMMOBILON™-P PVDF membrane (MILLIPORE®, Bedford,Mass.). After preblocking strips of the membrane in TBS containing 0.1%TWEEN®20 and 5% nonfat skim milk, the first antibody reaction wasperformed in the 1:2000 dilution of the affinity-purified antibodyagainst mSir2α or control rabbit IgG. The mSir2α band was visualizedwith the secondary anti-rabbit IgG antibody conjugated with horse radishperoxidase and the ECL detection kit (AMERSHAM PHARCIA BIOTECH INC.,Piscataway, N.J.). For immunoprecipitation, approximately 300 μg of theextract was incubated at 4° C. with 1 μg of the affinity-purified mSir2αantibody for 1 hr and then with Protein A-SEPHAROSE® beads (SIGMA®) foranother 1 hr. The protein complex on beads was washed with extractionbuffer three times and extracted in sample buffer. The protein waselectrophoresed and blotted as described above.

Immunofluorescence

NIH3T3 cells or mouse embryonic fibroblasts were plated on 10-wellmultitest slideglasses (ICN BIOMEDICALS, Aurora, Ohio). The cells werebriefly washed with PBS, fixed in PBS containing 3.2% paraformaldehydefor 10 min, and then treated with PBS containing 0.5% IGEPAL®CA-630 for20 min. Preblocking reaction was performed with PBG buffer (PBScontaining 0.2% cold water fish gelatin and 0.5% bovine serum albumin(SIGMA®) at 37° C. for 20 min. After washing cells in PBS-T (PBScontaining 0.2% TWEEN®20) briefly, the first antibody reaction wasperformed at 37° C. for 1 hr in PBG containing the affinity-purifiedmSir2α antibody diluted to 1:500 and/or one of the anti-human nuclearantibodies (ANA-N for nucleolus or ANA-C for centromeres, SIGMA®)diluted to 1:5 or 1:10 or control rabbit IgG. The cells were washed inPBS-T for 5 min twice. The secondary antibody reaction was performed at37° C. for 1 hr with the anti-rabbit IgG antibody conjugated with FITC(Jackson ImmunoResearch Laboratories, PA) and/or the anti-human IgGantibody conjugated with TEXAS-RED® (Vector Laboratories, CA). Afterwashing in PBS-T for 5 min, the cells were counterstained in 200 ng/mlof DAPI for 1 min. They were washed in TBS-T for 5 min again and in dH₂Obriefly, and then embedded under coverslips with VECTORSHIELD® (VECTORLABORATORIES).

For the immuno-FISH of mSir2α and telomeres, the first antibody reactionfor mSir2α, was performed as described above. The cells with the firstimmune complex was then fixed with 3.2% paraformaldehyde for 10 min,permealized again in PBS containing 0.5% CA630 IGEPAL®CA-630 for 20 min,treated in 0.1M NaOH for 2 min to denature DNA, and immediately washedin ice-cold PBS. The synthesis of a telomeric probe, hybridization ofthe probe and visualization of the hybridized spots were done asdescribed previously. The secondary antibody reaction to detect mSir2αwas also done during the last visualization step.

All immunofluorescent digital images were obtained with the NIKONECLIPSE® TE300 fluorescent microscope equipped with a CCD digital camera(HAMAMATSU PHOTONICS®, Japan) and the METAMORPH® Imaging System Software(UNIVERSAL IMAGING CORP., PA).

Production of Recombinant Proteins

The yeast SIR2 gene or the mSIR2α full-length cDNA was cloned intopET28a vector (NOVAGEN®). BL21 (DE3)- and BL21 (DE3) pLysS with an extracopy of arginine tRNA gene was transformed with the ySIR2 and mSIR2αplasmids, respectively. Each transformed bacterial clone was induced in1 mM IPTG at 37° C. for 1 hr. The induced 6×His-tagged proteins werepurified with Ni-NTA agarose under native condition (see FIG. 4A). TheN-terminal fragment of mSir2α was prepared in the same way. The controlelute was prepared from a bacterial clone carrying pET28a vector only.The recombinant proteins were aliquoted and kept at −70° C.

Site-directed mutagenesis of mSIR2α core domain was performed with theGENEEDITOR™ SYSTEM (PROMEGA®, Madison, Wis.) according to the procedureprovided by the manufacturer. The mutant recombinant proteins wereprepared in the same way as described above.

ADP-Ribosylation Assay

The typical reaction was performed in 50 or 100 μl of the buffercontaining 5 mM Tris-HCl [pH8.0 or 9.0], 4 mM MgCl₂, 0.2 mM DTT, 1 μMcold β-NAD⁺ (SIGMA®) and 8 μCi nicotinamide adenine dinucleotide5′[α-³²P]triphosphate (NAD) as a donor of ADP-ribose (˜1000 Ci/mmol,AMERSHAM PHARMACIA BIOTECH INC.)). To check the sensitivity to poly- ormono-ADP-ribosylation inhibitors, 3-aminobenzamide (ICN PHARMACEUTICALS,INC., CA), benzamide, novobiocin and coumermycin A1 (SIGMA®) were addedto the reactions prior to adding Sir2 proteins, respectively. The wildtype or mutant recombinant proteins (0.5-1 μg for ySir2p and 10 μg ofmSir2α) were added with 4 μg of each histone purchased from ROCHEMOLECULAR SYSTEMS, INC. (Indianapolis, Ind.). For the peptideexperiments, 5-10 μg of unacetylated or acetylated N-terminal tailpeptides (amino acids 1-20) of H3 and H4 purchased from UPSTATEBIOTECHNOLOGY, INC. (Lake Placid, N.Y.) were used as substrates of theyeast and mouse Sir2 recombinant proteins.

To detect the activity of the endogenous mSir2α, which was usuallyimmunoprecipitated from 300-400 μg of the NIH3T3 whole cell extract, thereaction buffer and substrates were added directly to the immune complexon Protein A-SEPHAROSE® beads. The reaction mixture was incubated atroom temperature for 1 hr. For the reaction of snake venomphosphodiesterase (ROCHE MOLECULAR SYSTEMS, INC.), 1-2 μl of the enzymeor the same volume of dH₂O was added to the reaction mixture,respectively, after finishing ADP-ribosylation reaction. Each mixturewas incubated at 37° C. for 1 hr and the reaction was terminated asdescribed below.

To analyze histone modification, 25 μl of 100% trichloroacetic acid(TCA) solution was mixed to the reaction mixture and left on ice for 15min. The protein precipitates were centrifuged at 4° C. and washed with5 or 20% TCA solution twice. The pellets were dissolved in 15 μl ofLeammli's sample buffer and boiled for 1.5 min. The proteins wereelectrophoresed in a 10-20% gradient SDS-PAGE gel. The gel was stainedwith COOMASSIE® Brilliant Blue R-250 (GIBCO BRL LIFE TECHNOLOGIES™,Frederick, Md.) to check equal loading of histones, dried, and exposedto KODAK® X-OMAT film.

To analyze modified peptides, 10 μl of each reaction mixture was spottedon a cellulose TLC plate (EM SCIENCE, Gibbstown, N.J.). Thechromatography was performed for 9-10 hrs in a TLC chamber containingthe 65:5:3:2:29 mixture of isobutyric acid, pyridine, acetic acid,butanol and water. The plate was dried and exposed to KODAK® X-OMATfilm. The peptide spots were visualized by ninhydrin staining.

Transient Transfection Assay

Effector plasmids of mSIR2α were constructed using DNA fragmentscorresponding to amino acids 220-500 of the wild type and mutant mSIR2αamplified by PCR with PFUTURBO® DNA polymerase (STRATAGENE®, La Jolla,Calif.) with primers that create EcoRI sites at both ends of eachfragment. Fragments were cloned into the EcoRI site of pM mammalianexpression vector (CLONTECH™) to produce the N-terminal fusion proteinto the GAL4 DNA binding domain. The luciferase plasmid, pUAS₄tkluc,which has four GAL4 binding sites in the upstream of the luciferasegene, was employed as a reporter gene. Plasmids preparations were madeusing the QIAFILTER™ Plasmid Midi kit (QIAGEN®).

NIH3T3 cells (10⁶ cells) were plated a day before transfection. Cellswere transfected using SUPERFECT™ transfection reagent (QIAGEN®) with 4μg of the GAL4DBD-core domain effector plasmid, 1 μg of the luciferasereporter gene and 1.5 μg of SV40 promoter-driven β-galactosidase gene tonormalize the transfection efficiency. The extracts of the transfectantswere prepared 48 hrs after transfection. The luciferase assay wasperformed using the luciferase assay system kit (PROMEGA®) and theOptocomp I luminometer (GM INSTRUMENTS™, CT), according to theprocedures provided by the manufacturers.

Production of Recombinant Proteins

The yeast SIR2 gene or the mSIR2α full-length cDNA were cloned intopET28a vector (NOVAGEN®, WI). BL21 (DE3) and BL21 (DE3) pLysS with anextra copy of arginine tRNA gene was transformed with the SIR2 andmSIR2α plasmids, respectively. Each transformed bacterial clone wasinduced in 1 mM IPTG at 37° C. for 1 hr. The induced 6×His-taggedproteins were purified with Ni-NTA agarose under native conditions. Thecontrol elute was prepared from a bacterial clone carrying pET28a vectoronly. The recombinant proteins were aliquoted and kept at −70° C.

Deacetylation and ADP-Ribosylation Assays

The typical reaction of Sir2 deacetylase activity was performed in 50 μlof buffer containing 50 mM Tris-HCl [pH 9.0], 4 mM MgCl₂, 0.2 mM DTT,variable concentration of cold nicotinamide adenine dinucleotide (NAD)or NAD derivatives (SIGMA®, MO), 5-10 μg of the purified recombinantSir2 proteins, and 10 μg of the histone H3 N-terminal tail peptide(amino acid 1-20) di-acetylated at positions 9 and 14 (UPSTATEBIOTECHNOLOGY, INC., NY). This starting peptide material contains acontaminant with 100 Da smaller molecular weight, which also showedexactly the same patterns of deacetylation (data not shown). To detectthe ADP-ribosylation activity, 8 μCi of NAD 5′-[α-³²P]triphosphate(˜1000 Ci/mmol, AMERSHAM PHARMACIA BIOTECH INC., Piscataway, N.J.) wasadded to the same reaction containing 1 μM cold NAD. Histone H3 protein(4 μg) (ROCHE MOLECULAR SYSTEMS, INC., IN) were used for this assay. Allreaction mixtures were incubated at room temperature for 1 hr.Trichostatin A and coumermycin A1 (SIGMA®) were prepared indimethylsulfoxide (DMSO, SIGMA®), and 5 μl of solvent or inhibitor wasadded to the reactions prior to adding Sir2 proteins.

Analysis of Deacetylated or ADP-Ribosylated Products

After the incubation, the products were precipitated at −20° C.overnight by adding 50 μl of distilled water and 25 μl of 100%trichloroacetic acid (TCA) solution. For high pressure liquidchromatography (HPLC), the precipitates were reconstituted in 5% CH₃CNand 0.1% trifluoroacetic acid (TFA) and run in the gradientconcentration between 0.05% TFA and 0.043% TFA plus 80% CH₃CN on HEWLETTPACKARD® Model 1100 HPLC System with 214TP52 column (VYDAC®, CA). Thechromatograms at the absorbance of 210 nm were digitally recorded andanalyzed by HEWLETT PACKARD® CHEMSTATION® System (version A.06.03[509]).Fractions of samples were collected every 1 min by GILSON® FractionCollector Model 203. Peptide sequencing was done by the APPLIEDBIOSYSTEMS® PROCISE® 494 HT Protein Sequencing System. PTH amino acidchromatograms were recorded and analyzed by the ABI Model 610A2.1™ dataintegration/analysis system.

Electron-spray mass spectroscopy (also referred to herein as massspectroscopy) was done on the PE Sciex Model API365™ system. Matrixassisted laser desorption/ionization (MALDI) mass spectroscopy can alsobe used. The electron-spray mass spectroscopy data were analyzed by theBioMultiView Program™ (version 1.3.1). To detect the ADP-ribosylatedintact H3 protein, the pellets were dissolved in 15 μl of Leammli samplebuffer, boiled for 1.5 min and electrophoresed in a 10-20% gradientSDS-PAGE gel. The gel was stained with COOMASSIE® Brilliant Blue tocheck equal loading of histones, dried, and exposed to KODAK® X-OMATfilm. To analyze the ADP-ribosylated H3 peptides, 10 μl of each reactionmixture was spotted on a cellulose TLC plate (EM SCIENCE, NJ). Thechromatography was performed for 9-10 hrs in a TLC chamber containingthe 65:5:3:2:29 mixture of isobutyric acid, pyridine, acetic acid,butanol and water (Scheidtmann, K. H., et al., J. Virol. 44:116-133(1982)). The plate was dried and exposed to KODAK® X-OMAT film. Thepeptide spots were checked by ninhydrin staining.

Molecular Cloning of mSIR2α

The mouse 15-day embryo 5-STRETCH PLUS™ cDNA library (CLONTECH™, CA) wasscreened with the EST cDNA fragment of AA199012 as a probe. Fivepositive clones were obtained from approximately one million independentplaques. One of the five clones contained a 3.9 kb cDNA fragment. Thenucleotide sequence of this fragment was determined with an APPLIEDBIOSYSTEMS® 374 Automated Sequencer. The isolated cDNA clone encodes thefull-length mSir2α protein since the in vitro-translated protein fromthis cDNA clone showed an indistinguishable size (110-120 kD) to theprotein in the mouse NIH3T3 extract recognized by a specific polyclonalantibody against a N-terminal portion of mSir2α (data not shown).

The amino acid sequences of mSir2α and other Sir2 family members werealigned in the CLUSTAL X program and a phylogenetic tree was generatedby using the NJPLOT program.

Strains, Plasmids, and Antibodies

All strains used were derivatives of W303a sir2AE: W303R sir2AE(MATa,ade2-1, leu2-3, 112, trp1-1, ura3-52, his3-11, sir2::TRP1, rDNA-ADE2),W303RT sir2AE(MATa, ade2-1, leu2-3, 112, trp1-1, ura3-52, his3-11,rad5-535, sir2::TRP1, rDNA-ADE2, URA3-TEL???), and W303Rsir2AE/rpd3AE(MATa, ade2-1, leu2-3, 112, trp1-1, ura3-52, his 3-11,rdp3::URA3, sir2::TRP1, rDNA-ADE2). Two integrating plasmids thatcontain SIR2 driven by its native promoter were used: pRS305-SIR2 andpRS305-SIR2*, the latter expressing Sir2p at low levels. SIR2 and mutantsir2 strains were generated by cutting the plasmid within the LEU2 geneand integrated using standard yeast transformation protocols. Unlessotherwise noted derivative strains were generated using pRS305-SIR2.Rabbit antibodies to Sir2p were generated by using full-length rSir2pisolated under denaturing conditions.

Generation of Core Domain Mutants

Site directed mutations were generated in the plasmid pRS305-SIR2* usingthe GENEEDITOR™ SYSTEM (PROMEGA®, Madison, Wis.) according to theprocedure provided by the manufacturer. Sequences were verified bySanger sequencing methods. The mutants were then subcloned intopRS305-SIR2 and pET28a.

ADP-Ribosylation and Deacetylation Assays

BL21 with SIR2 or the sir2 mutants subcloned into pET28a (NOVAGEN®) wereinduced with 1 mM IPTG for 1 hr. The recombinant proteins were purifiedby Nickel-NTA column under native condition. Ribosylation anddeacetylation assays were performed using 1 μg of recombinant proteinfor the ribosylation assay and 5 μg for the deacetylation assay.

Silencing, Life Span and rDNA Recombination Assays

To evaluate silencing at the telomeres and rDNA, 10-fold dilutions ofthe derivatives of either W303RT or W303R AErpd3 were spotted on mediacontaining 5-FOA or media lacking adenine, respectively. To assay for HMsilencing, W303R derivatives generated with pRS305-SIR2* were patchedonto YPD with the tester strain CKy20(MATa, arg1, tsm11) and afterovernight growth were replica plated to minimal media with nosupplemented amino acids. Life span and rDNA recombination rates weremeasured as in Kaeberlein, et. al., (Kaeberlein, M., et al., Genes Dev.13: 2570-2580 (1999)).

Results

Characterization of the Closest Mouse SIR2 Homolog, mSIR2α

At least three different sequences, mSir2α, β, and γ, related to yeastSir2p (ySir2p) have been identified in the mouse EST database. mSir2αwas identified as having the greatest homology to ySir2p and the relatedyeast protein, Hst1p (FIG. 1A). mSir2β and γ have significant homologyto Hst2p, another member of yeast Sir2 family.

The tissue distribution of mSIR2α, β, and γ mRNA expression was examined(FIG. 1B). A major transcript encoding mSIR2α (˜4.0 kb) is present inall tissues examined. Minor transcripts of various size are alsodetected in each tissue. mSIR2β has only one transcript (˜1.9 kb).mSIR2α and β appear to be expressed ubiquitously, showing a high levelof expression in liver. The mSIR2γ transcript (˜1.6 kb) is alsopredominantly expressed in liver.

A 3.9 kb cDNA clone of mSIR2α was identified by screening a mouseembryonic cDNA library with the EST clone AA199012 as a probe. This cDNAclone encodes a predicted protein with 737 amino acids with a molecularweight of 80.3 kD. The deduced amino acid sequence of mSIR2α is shown inFIG. 2A. A predicted nuclear localization signal KRKKRK (SEQ ID NO: 29)is present.

A rabbit polyclonal antibody raised against the N-terminal 131 aminoacids of the mSir2α protein recognized a 120 kD protein in the cellextracts of murine NIH3T3 cells (FIG. 2B) and embryonic fibroblasts(data not shown). The in vitro translated (IVT) protein from the cDNAclone has an indistinguishable size to the protein in the NIH3T3extract, suggesting that this cDNA clone encodes a full length proteinof mSir2α (right panel, FIG. 2B). The 120 kD band was not detected inimmunoprecipitates incubated with rabbit IgG alone or the in vitrotranslation mixture containing no RNA (FIG. 2B). An asterisk indicatesIgG in immunoprecipitates.

In the mSir2α, the greatest homology to ySir2p resides in the middle orcore domain of the protein (FIG. 2C). The amino acid identity of themSir2α core domain to ySir2p is 45.9% and the N-terminal region of themSir2α protein also shows weak homology to ySir2p (13.4% identity). ThemSir2α has a longer C-terminal region than ySir2p, but no significanthomology to other proteins in this region. The highly conserved coredomains of yeast and mouse SIR2 family members have homology to theSalmonella typhimurium CobB protein (Tsang, A. W., et al., J. Biol.Chem. 273: 31788-31794 (1998)) (20.3% identity in the core domain (FIG.2D). Asterisks denote a motif of putative NM) binding clefts conservedin known mono-ADP-ribosyltransferases. (FIG. 2C).

In yeast, immunostaining revealed Sir2p localization in the nucleolusand telomeres (Gotta, M., et al., Embo J. 16: 3243-3255 (1997)).Immunostaining of mouse NIH3T3 cells with a polyclonal antibody raisedagainst the N-terminal fragment of mSir2α localized mSir2 cc in thenucleus (FIG. 3A). DAPI-dense regions in nuclei, which correspond tomouse centromeres, were excluded (FIGS. 3B and C). This staining patternwas observed throughout the cell cycle except on mitotic chromosomes,which lacked staining (data not shown). Surprisingly, mSir2α was notlocalized to nucleoli, telomeres, or centromeres byco-immunofluorescence (FIG. 3, D-F, G-I, J-L) indicating that is notassociated with the most highly repeated DNA in the mouse genome.

Yeast and Mouse Sir2 Proteins are Novel NuclearMono-ADP-Ribosyltransferases

The ADP-ribosylase activity of bacterial recombinant full-lengthproteins of ySir2p and mSir2α was determined in in vitro assays (FIG.4A). Calf thymus histones were used as substrates and ³²P-labelednicotinamide adenine dinucleotide (NAD) as a donor of ADP-ribose.Reaction products were visualized by SDS-PAGE. The recombinant rySir2pand r-mSir2α proteins transferred radioactive ADP-ribose to H2B and H3,but not to H1, H2A and H4 (FIG. 4B, C). The Sir2 proteins modified H2Band H3 in a dose-dependent manner, the ADP-ribosylase activity did notreside in the mSir2α-specific N-terminal fragment N-ter (FIG. 4D). Asshown in the upper panel of FIG. 4D, 5, 10, and 15 μg of recombinantfull-length mSir2α (r-mSir2α) or its N-terminal fragment (N-ter) orcontrol eluate (pET) were added to the reaction with histone H2B. Onlythe full-length protein showed the dose-dependent modification. As shownin the lower panel of FIG. 4D, 100, 250, 500, and 1000 ng of recombinantfull-length ySir2p (r-ySir2p) were added to the reaction with histoneH3. ADP-ribosylase activity was not observed when histone proteins wereincubated with control pET alone (FIG. 4B).

To gain further evidence that this modification is due to ADP-ribose,the ySir2p- or mSir2α-modified H2B and H3 with snake venomphosphodiesterase (SVP) that can digest the phosphodiester bond in theADP-ribose moiety (FIG. 4C, E). The radiolabeled P³² (P*) is located ata position and the phosphodiester bond between radiolabeled andnonlabeled phosphates can be digested by snake venom phosphodiesterase(SVP), resulting in the removal of the radiolabel from ADP-ribosylatedproteins (FIG. 4E). The radiolabel could be substantially removed fromH2B and H3 by SVP (FIG. 4E). The label remaining in the histones may bedue to the forward reaction by Sir2 enzymes and NAD during thistreatment.

Histone products of Sir2 modification of unique molecular weights wereobserved in this assay, suggesting that this modification may not bepoly-ADP-ribosylation, but mono-ADP-ribosylation. The sensitivity of theADP-ribosylase activity of mSir2α to poly ADP-ribosylation inhibitors(3-aminobenzamide and benzamide) and mono-ADP-ribosylation inhibitors(novobiocin and coumermycin A1) was evaluated. (FIG. 4F).3-aminobenzamide and benzamide did not inhibit the ADP-ribosylationactivity of the mSir2α protein at concentrations of 40, 200 and 1000 μM(FIG. 4F, lanes 2 to 7) whereas novobiocin and coumermycin A1 stronglyinhibited ADP-ribosylation at concentrations of 200 μM and 40 μM,respectively (FIG. 4F, lanes 9 and 12), consistent with their reportedIC₅₀ concentrations of 370 and 27 μM. ySir2p showed the same sensitivityto those inhibitors (data not shown). Therefore, the sensitivity of theSir2 enzymatic activities to those inhibitors demonstrates that theseproteins are of the mono-ADP-ribosyltransferase class.

ADP-ribosyl-transferase activity could be detected for the endogenousmSir2α immunoprecipitated from NIH3T3 whole cell extracts (FIG. 4G). Theendogenous mSir2α was immunoprecipitated from 300 ng of the NIH3T3 wholecell extract with an anti-mSir2α polyclonal antibody and the immunecomplex on Protein A SEPHAROSE beads was incubated in the reactionbuffer containing ³²P-labeled NAD and histone H1 or H2B.

ADP-Ribosyltransferase and NAD Hydrolase Activities of Yeast and MouseSir2 are Specifically Triggered by Acetylated H3 and H4N-Terminal TailPeptides

Extended N-terminal tails of H3 and H4 are known to be important for theformation of Sir complex-mediated heterochromatin structure in yeast(Hecht, A., et al., Cell 80: 583-592 (1995)) and are targets for severaldifferent types of histone modification, such as acetylation,phosphorylation, and methylation (Hansen, J. C., et al., Biochemistry37: 17637-17641 (1998)). The residues 1-20 of H3 are conserved throughevolution and lysines 9 and 14 which are acetylated in the diacetylatedpeptide are indicated by asterisks in FIG. 5A. The residues 1-20 ofmonoacetylated H4 peptide originate from the human sequence andacetylated residues are indicated by asterisks FIG. 5A. Tetra-acetylatedH4 peptide corresponds to the Tetrahymena sequence where all lysines areacetylated. The N-terminal tails of histones were evaluated as targetsfor ADP-ribosylation by Sir2 proteins.

Two synthetic peptides corresponding to the first 20 amino acids of H3either unacetylated or diacetylated (on lysines 9 and 14) (FIG. 5A) wereevaluated to determine whether yeast and mouse Sir2 proteins couldmodify these peptides. Reactions were carried out as above and analyzedby thin layer chromatography. Both the yeast and mouse Sir2 modifiedonly the diacetylated H3 tail peptide (FIG. 5B). The indicated spot(bracket, FIG. 5B) was identified as the modified peptide because itmigrated just below the ninhydrin-positive peptide spot on thechromatogram. Further, NAD hydrolase activity of both ySir2p and mSir2αwas stimulated in the presence of the diacetylated peptide, indicated bythe arrowhead in FIG. 5B. Since Lys9 and Lys14 of H3 are acetylated invivo, this preferential utilization of the acetylated H3 peptidestrongly suggests that the tail of H3 is a biologically relevantsubstrate for Sir2 proteins.

The extended N-terminal tail of histone H4 has four lysines at positions5, 8, 12, and 16. (FIG. 5A). Mutational studies show the importance ofthese residues in silencing in vivo, and Lys5, 8, and 16 arehypoacetylated in yeast chromatin at HM loci. In mammalian cells, Lys16of H4 is highly acetylated. H4 peptides (residues 1-20), which wereacetylated singly at each of these lysines (5, 8, 12 or 16), weresynthesized and evaluated for mSir2α stimulated ADP-ribosyltransferaseand NAD hydrolase activities (FIG. 5C). A striking specificity in theacetylation pattern of the H4 tail was required to stimulate Sir2activity. Both ADP-ribosylation and NAD hydrolysis were stimulated bythe Lys16Ac peptide, but were affected weakly by the Lys5-, 8-, 12-, andtetra-acetylated peptides (FIG. 5C).

Highly Conserved Amino Acid Residues in the Core Domain are Essentialfor the Mono-ADP-Ribosylation Activity of mSir2α

To analyze the mono-ADP-ribosylation activity more precisely, ten highlyconserved amino acid residues in the core domain of mSir2α were changedto alanine (FIG. 6A), mutant recombinant Sir2 proteins produced E. coli,and evaluated for ADP-ribosylation activities on H2B and H3 in vitro.Protocols for the production of mutant nucleic acid constructs andproteins are well known to the skilled artisan. (See, for example,Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley &Sons, NY, N.Y. (1999)).

Aliquots of recombinant protein (10 μg) of the wild type recombinantmSir2α (WT), the pET control pET, and the mutant proteins (G253A, G255A,S257A, I262A, F265A, R266A, G270A, P285A, T336A, H355A) were incubatedwith H2B or H3. Western blot analysis showed that the amount of eachfull-length protein used was comparable (FIG. 5B, r-mSirα). All of themutations of highly conserved residues dramatically affected themono-ADP-ribosylation activity of mSir2α (FIG. 6B). The results weresimilar using H2B or H3.

Eight mutants (G253A, G255A, S257A, I262A, F265A, G270A, T336A, H355A)completely abolished the activity and the two mutants R266A and P285Ashowed some residual activities compared to the wild type activity (18%for R266A and 5% for P285A, see FIG. 6C). The radioactivity of H2Bmodified by the wild type is assigned to 100% and other data werenormalized according to the wild type activity and blots quantitated byPhosphorimaging. The averages and the standard deviations werecalculated from two independent experiments. In the core domain ofySir2p, mutations on highly conserved residues also affected themono-ADP-ribosylation activity. Therefore, the core domain of Sir2proteins contain the catalytic activity of mono-ADP-ribosylation and theevolutionarily conserved residues in the core domain are essential forthis activity.

The mSir2α Core Domain Conveys Transcriptional Repressive Activity inMouse Cells

The in vivo function of the mSir2α mono-ADP-ribosylation activity, wasevaluated by constructing expression vectors of wild-type (WT) and eachof the mutant core domains (G253A, G255A, S257A, I262A, F265A, R266A,G270A, P285A, T336A, H355A) fused to the yeast Gal4 DNA binding domain(DBD) and transfecting the constructed NIH3T3 cells with a luciferasereporter plasmid containing four upstream Gal4 binding sites.

NIH3T3 cells 10⁶ were transfected with 4 μg of effector plasmids whichfuse Sir2 sequences to the DBD, 1 μg of a reporter plasmid withluciferase expression driven by GAL4 DNA-binding sites and 1.5 μg ofSV40 promoter-driven β-galactosidase gene to normalize the transfectionefficiency. All luciferase activities after normalization werestandardized to DBD alone. The averages and the standard deviations werecalculated from three independent transfection experiments. Thewild-type core domain was able to repress transcription four-fold,compared to the activity of the DBD only, which is known to activatetranscription approximately two-fold in mammalian cells (FIG. 6D). TheDBD-fused full-length ySir2p also showed similar repressive activity inNIH3T3 cells, suggesting that the shared mono-ADP-ribosylation activitycan be involved in transcriptional repressive activity. All of the coredomain mutants except for P285A reduced transcriptional repressiveactivity (FIG. 6D). One explanation for this discrepancy is that theP285A change does not inhibit the catalytic domain but causes therecombinant protein to be misfolded in E. coli. Another protein thatbinds to mSir2α can rescue such a mutant in mammalian cells.

Sir2p is an NAD-Dependent Deacetylase for Histone H3

The experiments employed purified recombinant Sir2p in a reaction withNAD and a peptide of the histone H3 amino-terminal tail (residues 1-20)di-acetylated at lysines 9 and 14. These lysines are hypoacetylated insilenced chromatin and mutations at these positions to glycine greatlyreduce silencing in vivo (Thompson, J. S., et al., Nature 369: 245-247(1994)). The products of a reaction containing 5 μg of recombinant yeastSir2p (79 pmoles) (FIG. 8 a), 10 μg of the H3 peptide (4.2 nmoles)increasing concentrations of NAD were analyzed s by high pressure liquidchromatography (HPLC).

As shown in FIG. 8 b, the histone peptide reacted in the absence of NADgave rise to two peaks (3 and 5) which were analyzed by electron-spraymass spectroscopy (FIGS. 9 a and 9 d) and correspond to monomer (MW2370)and dimer (MW4740) peptide, the latter likely due to oxidation of thepeptide at the carboxyl cysteine residue. The same species were observedin reactions with a control bacterial preparation (“pET” in FIG. 8 a) inthe presence of NAD (not shown).

The addition of NAD to the reaction containing Sir2p gave rise to threeadditional peaks (1, 2, and 4), as well as an alteration in peak 3(FIGS. 8 c, 8 d, 8 e and 8 f), which were also analyzed byelectron-spray mass spectroscopy. Strikingly, these peaks did notcorrespond to ADP-ribosylated species, but rather to deacetylatedspecies of peptide (see FIG. 8 g). Peak 4 corresponded to thesingly-deacetylated dimer (MW 4698) (FIG. 9 c), peak 3 now alsocontained the doubly-deacetylated dimer (MW4656) (FIG. 9 b). Because oftheir lower abundance, peak 1 and peak 2 were analyzed separately bymatrix assisted laser desorption/ionization (MALDI) mass spectroscopyand peak 2 was found to correspond to the triply deacetylated dimer(MW4614) and peak 1 to the singly-deacetylated monomer (MW2328) (notshown). The relative areas under peaks 1, 2, and 4 was quantitated andat least 27% of the input peptide was deacetylated by Sir2p. Theapproximate Km of this deacetylation reaction for NAD was about 100 μM,at which the reaction proceeded to about 50% of the maximal levelobserved at higher concentrations of the cofactor.

To analyze these reaction products further, peak 4, the singlydeacetylated dimer, along with the input peak 5 were subjected toN-terminal protein sequencing by Edmann degradation (FIG. 10). The onlydifferences between the input and reacted peaks occurred at lysines 9and 14. Approximately 23-27% of the acetyl lysines at each position weredeacetylated by Sir2p in the presence of NAD, in agreement with thecalculated efficiency of the reaction and the mass spectroscopy dataabove. The unacetylated lysine 18 of peaks 4 and 5 is also shown forcomparison. Thus, Sir2p is an NAD-dependent histone deacetylase whichcan deacetylate either lysine 9 or 14 of the H3N-terminus.

Effects of Inhibitors on Deacetylation and Putative ADP-Ribosylation

The effects of a potent inhibitor of histone deacetylases, trichostatinA (TSA) (Yoshida, M., et al., A. J. Biol. Chem. 265: 17174-17179 (1990))was evaluated. TSA has been shown to act on constitutive heterochromatinin vivo, since it disrupts centromeric heterochromatin in S. pombe(Ekwall, K., et al., Cell 91: 1021-1032 (1997)). As shown in FIGS. 11 aand 11 b, TSA was totally incapable of inhibiting deacetylation,indicating that Sir2p is fundamentally different from the class ofTSA-sensitive histone deacetylases, including Rpd3 (Taunton, J., et al.,Science 272: 408-411 (1996)).

Sir2 proteins have been proposed to transfer a single ADP-ribose fromNAD to protein (Frye, R. A., Biochem. Biophys. Res. Commun. 260: 273-279(1999)). Consistent with this, we found that ³²P was transferred fromNAD to intact histone H3 (FIG. 11 c) or to the H3 peptide (FIG. 11 d).Transfer of label to the peptide was assayed using thin layerchromatography (TLC), which revealed not only this transfer but asubstantial amount of NAD hydrolysis.

The effects of a known inhibitor of mono-ADP-ribosyltransferases,coumermycin A1 (Banasik, M., et al., Mol. Cell. Biochem. 138: 185-187(1994)) was evaluated. Both ADP-ribosylation (FIGS. 11 c and 11 d) andNAD hydrolysis (FIG. 11 d) were inhibited by this drug at concentrationsknown to inhibit other mono-ADP-ribosyltransferases. Strikingly,coumermycin A1 was not capable of inhibiting deacetylation of the H3tail by Sir2p (FIG. 11 e). Thus, the ADP-ribosyltransferase and NADhydrolase reactions are fundamentally distinct from this NAD-dependentdeacetylation reaction. These two separable enzymatic activities ofSir2p may play distinct roles in vivo.

Mouse Sir2p Also Catalyzes the Deacetylation of the Histone H3 Tail

A mouse Sir2 homolog was identified in the EST database and cloned thefull length cDNA, termed mSir2α (FIG. 12 a). The conserved region ofthis protein resembles yeast Sir2p more closely than do other mouse Sir2proteins (FIG. 12 b). To determine whether the mouse Sir2p homologmSir2α would catalyze this deacetylation reaction, we incubated purifiedrecombinant mSir2α with the di-acetylated H3 peptide and analyzed thereaction products by HPLC (FIG. 12 c). The murine protein gave rise tothe same array of products with a similar yield as the yeast enzyme,indicating that this deacetylation reaction is conserved from yeast tomammals.

NADH, NADP, and NADPH do not Activate Deacetylation by Sir2p

While there are literally hundreds of NAD-linked dehydrogenases that useNAD and NADH in oxidation/reduction reactions in cells, for example,glyceraldehyde-3-phosphate dehydrogenase, the requirement for NAD todrive any other enzymatic reaction is novel. This suggested that Sir2proteins might be sensors of the energy or oxidation state of cells thattransduces this status to the organization of chromatin structure.Catabolic reactions in cells, such as substrate level and oxidativephosphorylation in the utilization of glucose, are oxidative and use NADto produce NADH. Biosynthetic reactions typically are reductive and useNADPH to produce NADP. It was thus of interest to determine whetherNADH, NADP, and NADPH would function in the deacetylation reaction.

As shown in FIG. 13, NADP and NADPH did not function significantly,while NADH functioned weakly in the deacetylation of the H3 peptide bySir2p. In fact, the small amount of deacetylated products in the NADHreaction can reasonably be attributed to a low level of oxidation of theNADH preparation. Also, neither NADH nor NADP significantly inhibitedthe deacetylase activity in a reaction with NAD (not shown). Thisremarkable specificity of Sir2p for NAD but not the other dinucleotidesmay allow Sir2 proteins to sense the energy or oxidation status of cellsto link histone deacetylation and chromatin silencing to the metabolicrate.

Histone Deacetylase Defective Sir2p Mutants Show Defects in Silencing,Recombination Suppression and Life Span

The present invention shows that mutations in Sir2 proteins reduce oreliminate the enzymatic activity of the protein affect these variousfunctions, indicating that the histone deacetylase activity of Sir2p isimportant in vivo.

A set of mutations in highly conserved residues of the core domain ofSIR2 were constructed by site-directed mutagenesis (FIG. 17 a) andcloned into vectors, along with the wild type, to allow expression ofthe recombinant proteins in E. coli or expression of single-copy genesfrom the native SIR2 promoter in S. cerevisiae. These 6× his taggedproteins were purified from E. coli by a Ni-NTA column (FIG. 17 b) andanalyzed for the NAD-dependent histone H3 deacetylase activity in anassay with a di-acetylated H3 peptide (residues 1-20 acetylated on Lys9and Lys14) and 1 mM NAD. HPLC separation of the reaction products yieldsfive peaks of which 1, 2, a portion of 3 and 4 are deacetylated speciesof peptide (FIG. 15). In this deacetylase assay, mutants 345 and 347were inactive, mutant 261 showed 17% of wild type activity, mutants 270and 271 showed 80% and 36% wild type activity, respectively, and mutant275 showed 67% wild type activity.

The Sir 2 mutant proteins were also analyzed for ADP-ribosyltransferaseactivity using the histone H3 substrate, (FIG. 17 c). Mutants 345 and347 were completely inactive, mutants 261, 270, and 271 displayed veryweak activity, 4%, 7%, and 8%, respectively, and mutant 275 was about asactive as wild type. The same pattern of activities was observed using apeptide of the N-terminal tail of histone H3 (not shown).

To examine the functions of these mutants in vivo, we studied yeastindicator strains in which the endogenous SIR2 had been deleted and thewild type and mutant SIR2 genes integrated back into the genome. Allstrains used are isogenic derivatives of W303R (Kaeberlein, M., et al.,Genes Dev. 13: 2570-2580 (1999)) and all results are summarized in FIG.18. The mutant proteins were visualized by Western blot and found to beexpressed at approximately the same level as wild type, with a smallreduction in the level of mutant 261 and perhaps 275, and 345 (FIG. 16a).

Silencing of the HMLα and HMRa mating loci was assayed by mating with ahaploid strain of opposite mating type and monitoring the appearance ofdiploids on selective media. In this assay (FIG. 16 b), mutants 261,270, and 271 were mating proficient, indicating that silencing wasessentially intact, and mutants 345, 347, and, surprisingly, 275 weredefective. Telomere silencing was determined by repression of thetelomere-positioned URA3 gene, which gives rise to growth on mediacontaining 5-fluoro-orotic acid (FOA). Serial dilution of wild type andmutant strains on FOA plates indicated that mutant 261 silenced about aswell as wild type, 270 showed partial silencing, and 271, 275, and 345′were defective (FIG. 16 c). Silencing in the rDNA was determined by aserial dilution onto adenine-lacking media of strains with ADE2 insertedinto the rDNA (Kaeberlein, M., et al., Genes Dev. 13: 2570-2580 (1999)).To facilitate detectable of differences in rDNA silencing, a rpd3AEstrain was used which displays enhanced silencing (Smith, J. S., et al.,Mol. Cell. Biol. 19: 3184-3197 (1999)). By this assay mutants 261 and270 silenced about as well as the wild type, mutant 271 showed weaksilencing, and mutants 275, and 345 were totally defective (FIG. 16 d).

The data indicate a general, although not strictly quantitative,correlation between deacetylase activity in vitro and silencing in vivo(except for 275, see below) Mutant 345 is inactive for deacetylaseactivity in vitro and silencing in vivo. Mutants 261, 270, and 271, haveintermediate levels of deacetylase activities in vitro and intermediateeffects on silencing in vivo. Mutant 261 is proficient in all silencingassays, but does show a defect in a more sensitive silencing assay oflife span, below. Mutant 270 is proficient in HM and rDNA silencing butintermediate in silencing at telomeres, and 271 is proficient in HMsilencing, but intermediate to weak in silencing at telomeres and rDNA.An intermediate levels of deacetylase activity can are sufficient tomediate silencing, but the specific level of silencing may be modulatedby other variables affected by these mutations, such as the interactionof Sir2p with partner proteins. The lack of silencing activity in mutant275 was unexpected and may reflect an inability of that mutant proteinto localize properly in the nucleus or to interact with importantpartners in vivo.

Replicative life span of mother cells is due in part to the accumulationof extrachromosomal rDNA circles (Sinclair, D. A., et al., Science 277:1313-1316 (1997); (Sinclair, D. A., et al., Cell 91: 1-20 (1997)) thatarise by homologous recombination (Park, P. U., et al., Mol. Cell. Biol.19: 3848-3856 (1999)) in the tandem array of rDNA repeats on chromosomeXII. This recombination is suppressed 5-10 fold by SIR2 (Gottlieb, S.,et al., Cell 56: 771-776 (1989)), allowing cells to enjoy their normallife span. To measure recombination frequencies, wild type and Sir2mutant strains containing the ADE2 gene inserted into the rDNA wereplated on YPD media. This media is limiting in adenine, causingade2-colonies to accumulate a red pigment. Half-red/white sectoredcolonies indicate ADE2 loss in the first generation after plating, andthe frequency of these colonies compared to Ade+ colonies is a directmeasure of the recombination rate in the rDNA. By this assay, SIR2suppressed recombination about 12-fold compared to the SIR2 deletionstrain (FIG. 17 a). Mutants 261 and 270, showed a high degree ofsuppression in the range of wild type, mutant 271 showed partialsuppression, and mutants 275, and 345 were as defective as the SIR2deletion. Thus, the activities of these mutants in this recombinationassay are similar to their activities in the rDNA silencing assay,above.

Previous findings show that the short life span of SIR2 deletion mutantsin strain W303R is a composite of a failure to suppress rDNArecombination and also a failure to repress transcription of the copiesof mating type information at HM loci yielding the a/α cell type(Kaeberlein, M., et al., Genes Dev. 13: 2570-2580 (1999)). Expression ofa/α leads to a higher rate of recombination in the rDNA. The life spansof representative mutants were first determined in strains in which HMLa was deleted to avoid any contribution of the a/α cell type if weakderepression occurred at those loci. The sir2 deletion shortened lifespan about 50% compared to wild type, as expected (FIG. 17 b). Mutants261 and 270 complemented the sir2 deletion to give a wild type life spanand mutant 275 was completely defective in complementing the life spandefect, consistent with the activities of these mutants in therecombination assay.

Life span determination in wild type HM strains is a very sensitiveknown assay for Sir2 activity in vivo, i.e. in this assay even SIR2/SIR2heterozygous diploids show a pronounced defect (Kaeberlein, M., et al.,Genes Dev. 13: 2570-2580 (1999)). Thus, the life spans of SIR2 mutantswere also determined in otherwise wild type haploid strains (FIG. 17 c).Now a partial defect was observed in the 261 and 270 mutants, which canbe attributed to a slight defect in the maintenance of repression at HMloci. Mutant 275 was again defective. Thus, with this sensitive assay,the 261 and 270 SIR2 mutants show defects, which parallel theirenzymatic defects in vitro. Mutant 275 is exceptional, as in the aboveassays.

Previous studies indicate that the acetylation state of lysines in theamino-terminal tails of histones H3 and H4 is crucial for silencing invivo (Braunstein, M., et al., Genes Dev 7: 592-604 (1993); (Braunstein,M., et al., Mol. Cell. Biol. 16: 4349-4356 (1996); (Turner, B. M., etal., Cell. 69: 408-411 (1992)). Lysines 9 and 14 of H3 and lysines 5, 8,and 16 of H4 are hypoacetylated in silenced chromatin and return totheir acetylated state in SIR mutants (Braunstein, M., et al., Genes Dev7: 592-604 (1993); (Braunstein, M., et al., Mol. Cell. Biol. 16:4349-4356 (1996)). Moreover, mutations in the lysines of the H3 tail,especially to uncharged residues, cause a loss of silencing (Thompson,J. S., et al., Nature 369: 245-247 (1994)). These data show that Sir2mutations which eliminate deacetylase activity show silencing defects invivo.

Taken together these findings suggest that the NAD-dependent histonedeacetylase activity of Sir2p sufficiently explains the SIR2 functionsof silencing, suppression of rDNA recombination, and life span extensionin vivo. H3 and H4 are acetylated as a prerequisite to their recognitionby chromatin assembly factors (Ma, X. J., et al., Proc. Nat. Acad. Sci.95: 6693-6698 (1998)). Sir2p can be a target to specific genomic sitesand initiates silencing by deacetylating H3 and, most likely, H4. It ispossible that the putative ADP-ribosyltransferase activity of Sir2p alsoplays some role in these in vivo functions.

These data show that histone deacetylation by Sir2p has an absoluterequirement for NAD. These data are striking and can couple chromatinsilencing to the energy status of cells. Caloric restriction (Weindruch,R. H., et al., J. Nutrit. 116: 641-654 (1986)) increases life span in awide variety of eukaryotic species. This regimen may increase availableNAD and trigger the activity of Sir2 proteins, leading to greatersilencing and a longer life span. Similarly normal aging can be caused,in part, by a decrease in the activity of Sir2 proteins, perhaps owingto a reduction in available NAD, causing a loss of silencing and thedeleterious alteration in the pattern of gene expression.

Mouse Sir2p homolog mSir2α has histone deacetylase activity that closelyresembles yeast Sir2p. The Sir2 proteins described herein and relatedmammalian Sir2 proteins (Brachmann, C. B., et al., Genes Dev. 9:2888-2902 (1995); (Frye, R. A., Biochem. Biophys. Res. Commun. 260:273-279 (1999)) could play regulated roles in chromatin structure, whichmight include changes in genome organization during cellulardifferentiation and in response to physiological stimuli, includingcaloric restriction. If aging were due, at least in part, to a loss ofsilencing, then interventions to increase the activity of Sir2 proteinscan slow aging in mammals.

What is claimed is:
 1. A method of NAD-dependent deacetylation of atleast one lysine residue in an acetylated protein, comprising: combiningthe acetylated protein, with an isolated Sir2 protein or a fragment of aSir2 protein in the presence of NAD and in the absence of otherdeacetylases, wherein the fragment of the Sir2 protein comprises anamino acid sequence selected from the group consisting of the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 38;and detecting NAD-dependent deacetylation of at least one lysine residuein the acetylated protein.
 2. The method of claim 1, wherein theacetylated protein is selected from the group consisting of an H2B, H3and H4 histone protein.
 3. The method of claim 2, wherein the lysineamino acid residue includes at least one of the 4^(th), 9^(th) and14^(th) lysine residue of the H3 histone protein of SEQ ID NO:
 6. 4. Themethod of claim 2, wherein the lysine amino acid residue includes atleast one of the 5^(th), 8^(th), 12^(th) and 16^(th) lysine residue ofthe H4 histone protein of SEQ ID NO:
 7. 5. The method of claim 1,wherein the Sir2 protein is a Sir2α protein.
 6. The method according toclaim 5, wherein the Sir2α protein comprises the amino acid sequence ofSEQ ID NO: 1, 9, 12, 19 and
 26. 7. The method according to claim 5,wherein the Sir2α protein comprises the Sir2α protein encoded by thenucleic acid sequence of SEQ ID NO:
 25. 8. The method of claim 1,wherein the Sir2 protein is a human Sir2 protein.
 9. The method of claim8, wherein the human Sir2 protein comprises SEQ ID NO:
 38. 10. Themethod of claim 1, wherein the Sir2 protein is a recombinant Sir2protein.
 11. The method of claim 1, wherein the acetylated protein is anuclear protein.
 12. The method of claim 1, wherein the Sir2 protein isan isolated nuclear protein.
 13. The method of claim 1, wherein thefragment of the Sir2 protein comprises the amino acid sequence of SEQ IDNO:
 9. 14. The method of claim 1, wherein the fragment of the Sir2protein comprises the amino acid sequence of SEQ ID NO:
 19. 15. Themethod of claim 1, wherein the fragment of the Sir2 protein comprisesthe amino acid sequence of SEQ ID NO:
 38. 16. The method of claim 1,wherein the fragment of the Sir2 protein is selected from the groupconsisting of SEQ ID NOS: 4 and 12.